High throughput mobility shift

ABSTRACT

The present invention provides novel microfluidic devices and methods for performing pulsed field mobility shift assays in microfluidic devices. In particular the devices and methods of the invention utilize differences between electrophoretic mobilities (e.g., as between reactants and products, especially in non-fluorogenic reactions) in order to separate the species and thus analyze the reaction.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of pending U.S. Utility patentapplication Ser. No. 10/421,642 filed Apr. 23, 2003, which claims thebenefit of U.S. Provisional Patent Application No. 60/375,538, filedApr. 24, 2002, which is incorporated herein by reference in its entiretyfor all purposes.

FIELD OF THE INVENTION

The present invention provides microfluidic devices and methods for thehigh throughput detection and characterization of interaction of atleast two molecules in a microfluidic device based upon the molecules'differing electrophoretic mobility.

BACKGROUND OF THE INVENTION

When carrying out processes such as chemical or biochemical analyses,assays, syntheses, or preparations, a large number of separatemanipulations are performed on the materials being processed. Thesemanipulations include the measuring, aliquotting, transferring,diluting, mixing, separating, detecting, and incubating of thematerials. Microfluidic technology miniaturizes these manipulations andintegrates them so that they can be executed within a singlemicrofluidic device. For example, pioneering microfluidic methods ofperforming biological assays in microfluidic systems have beendeveloped, such as those described in U.S. Pat. No. 5,942,443 entitled“High Throughput Screening Assay Systems in Microscale Fluidic Devices”by Parce et al. and in PCT Published Application Number WO 98/45481entitled “Closed Loop Biochemical Analyzers” by Knapp et al.

A problem of particular interest in numerous applications ofmicrofluidic devices is the detection, characterization, andquantification of reactions that cannot be conveniently monitored bytaking fluorogenic measurements. Such reactions include reactions inwhich there is no measurable change in fluorescence when a reactionproduct is formed. For example, kinase reactions have no easyfluorogenic means of quantification. Mobility shift assays inmicrofluidic devices were devised to help overcome this problem.Mobility shift assays are described in U.S. Pat. No. 6,524,790 entitled“Apparatus and Methods for Correcting for Variable Velocity inMicrofluidic Systems,” by Kopf-Sill et al.

The mobility shift assays currently carried out in microfluidic devicescould be improved by increasing the throughput of those assays, and byexpanding the applicability of those assays to reactions not compatiblewith existing mobility shift assays. The throughputs of some existingmobility shift assays are adversely affected by the long transit timesrequired to separate some molecules based upon their electrophoreticmobility. These long transit times can lead to increased thermaldispersion of the separated groups of molecules, as well ashydrodynamically induced dispersion when pressure driven flow is used.Dispersion can adversely affect the throughput rate of an assay anddecrease separation resolution. Accordingly, minimizing the transittimes in a mobility shift assay can increase the throughput and improvethe resolution of an assay.

A welcome addition to the art would be the enhanced ability to increasethe throughput and resolution of mobility shift assays by decreasing thetransit time. The present invention describes and provides these andother features by providing new methods and microfluidic devices thatmeet these and other goals.

SUMMARY OF THE INVENTION

The present invention provides methods, systems, kits, and devices fordetection and characterization of interactions of molecules in amicrofluidic device based upon the molecules' differing electrophoreticmobility in a pulsed electric field. Molecules to be assayed are flowedthrough one or more microchannels and subjected to a pulsed electricfield. The molecules are then detected and their interactions arecharacterized.

In one aspect, the invention comprises a method of detecting aninteraction between a first molecule and at least a second molecule in amicrofluidic device involving flowing a first molecule that has a firstelectrophoretic mobility and is labeled with a first label together withat least a second molecule that has a second electrophoretic mobilityand is optionally labeled with the same or a different label than thefirst molecule, applying a pulsed electric field through themicrochannel to separate the first and at least second molecules basedupon their electrophoretic mobilities, and detecting levels of label (orlevels of signal from labels) in the microchannel over a select periodof time to determine the interaction (if any) between the molecules. Insome embodiments the at least second molecule comprises a secondmolecule and at least a third molecule. Embodiments of the invention maybe employed to assay molecular interactions in which the first andsecond molecules interact to form the third molecule. These types ofmolecular interactions occur between receptors and ligands (where thethird molecule comprises the receptor-ligand pair), antibodies andantigens (where the third molecule is an antibody-antigen complex), twoat least partially complementary nucleic acid strands (where the thirdmolecule comprises the resulting double-stranded nucleic acid), andenzymes and substrates (where the third molecule comprises the product).In the case of enzyme/substrate interactions, the product may comprisesthe label(s) from the first (or in some situations the second) molecule,and also may comprise the first molecule with a changed electrophoreticmobility. In some embodiments of methods in accordance with theinvention, the at least second molecule may comprise a derivative formof the first molecule.

In some embodiments, the method of detecting an interaction between afirst molecule and at least a second molecule further involves at leasta fourth molecule that can be one or more of a reaction enhancer, areaction inhibitor, or a reaction competitor. Additionally, in someembodiments one or more of the molecules being assayed optionallychanges one or more of its size, charge, or electrophoretic mobilityduring or after the interaction of the molecules. The interaction of themolecules in the assays of embodiments of the invention optionallycomprise an enzymatic reaction or a binding reaction.

In methods in accordance with the invention, the flow of moleculesthrough one or more microchannels may be one or more of electrophoreticflow, electroosmotic flow, pressure based flow, wicking based flow, orhydrostatic pressure based flow. The flow of the molecules can also beoptimized to increase assay sensitivity by, for example, increasing ormaximizing the difference in a reaction parameter as compared betweenwhen the pulsed electric field is in a first state (e.g., on, or at afirst value) and in a second state (e.g., off, or at a second value).Flowing can also comprise incubating the molecules being assayed, suchas the first and second molecules, for a specific period of time. Inother embodiments, flowing the molecules comprises continuouslyinjecting a first molecule into the microchannel and intermittentlyinjecting the at least second molecule into the same microchannel. Theintermittent injection optionally comprises a length of time at least aslong as the period of a pulse in the electric field. In variousembodiments, the molecules to be interacted, typically the first andsecond molecules, are both continuously flowed into/through themicrochannel either concurrently or non-concurrently, or are bothintermittently flowed into/through the microchannel where the flows ofthe different molecules are completely or partially overlapping.

In various embodiments of the methods of the invention, the label of oneor more of the molecules in the assay comprises a fluorescent label, achemiluminescent label, or a radioactive label. Furthermore, detectingof the components of the pulsed field assay can optionally includedetermination of reaction kinetics between the first and at least secondmolecule.

In some embodiments of the methods of the invention, the electrophoreticmobility of the first molecule is greater than that of the secondmolecule, while in other embodiments, the electrophoretic mobility ofthe first molecule is less than that of the second molecule.

The application of a pulsed electric field in methods in accordance withthe invention optionally comprises a first state comprising applying anelectric field that produces a first specific voltage or electriccurrent through the microchannel for a first specific period of timefollowed by a second state comprising applying an electric field thatproduces a second specific voltage or electric current for a secondspecific period of time. Such first and second periods of time areoptionally equal and in some embodiments range from about 0.1, 0.2, 0.3,0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, or 20 seconds or more, or any length inbetween. Alternatively, in some embodiments such periods of time are notof equal length and can different by a factor of 1, 2, 3, 4, 5, 10, 20,25, 30, 35, 40, 45, 50 or more, or any non-integral factor in between.In exemplary embodiments, the magnitude of the pulsed fields thatproduce the first and second specific voltages or electric currents canrange from at least about 10 V/cm to about 3,000 V/cm or more, from atleast about 50 V/cm to about 2,000 V/cm or more, from at least about 100V/cm to at least about 1,000 V/cm or more, or from at least about 250V/cm to at least about 750 V/cm or more.

In embodiments wherein a first molecule is continuously injected intothe microchannel and a second or more molecule is intermittentlyinjected into the microchannel, the periods of time when the pulsedfield is at a first value and then at a second value are optionallyselectively set in relation to the periods of time the second moleculeis flowed into the microchannel, which comprises a third period of time.Such third period of time can optionally be from about 1 to about 100 ormore times longer than the first and/or second periods of time.Alternatively, the third period of time can be at least about 5 to atleast about 75, from at least about 10 to at least about 50, or from atleast about 25 to at least about 45 times longer than the first and/orsecond periods of time.

In some embodiments, the microchannel in which the pulsed field assay isperformed can optionally comprise a polymer gel, a microfabricatedbarrier, or a sieving matrix.

In some aspects, embodiments of the current invention comprise anintegrated system or microfluidic device for detecting an interactionbetween a first molecule and at least a second molecule. Embodiments ofsuch systems or devices include a body structure with at least a firstmicrochannel disposed therein, a source of a first labeled molecule witha first electrophoretic mobility fluidly coupled to the microchannel, asource of at least a second molecule with a second electrophoreticmobility fluidly coupled to the microchannel, a fluid direction systemthat controllably moves the first and second molecules through themicrochannel; a voltage regulator system that controllably applies apulsed electric field through the microchannel wherein the pulsed fieldseparates the molecules based upon their electrophoretic mobilities, adetector system to detect the level(s) of label(s) or the level ofsignal from label(s) in the microchannel over time, and system softwarecomprising logical instructions to determine the interaction between themolecules based upon the electrophoretic mobility of the molecules inthe pulsed electric field.

In various embodiments, the sources of the first and second moleculesmay comprise the same or different sources. Additionally, the floweffectuated by the fluid direction system optionally comprises one ormore of electrokinetic flow, pressure driven flow, wicking driven flow,and hydrostatic pressure driven flow. Such flow optionally includes acontinuous flow of the first molecule and an intermittent flow of thesecond molecule; a continuous flow of both molecules; an intermittentflow of both molecules; or an intermittent flow of the first moleculeand a continuous flow of the second molecule. Additionally, the fluiddirection system optionally optimizes assay sensitivity by maximizing orincreasing the difference in one or more reaction parameters where suchparameters are compared between when the pulsed field is in a firststate and when the pulsed field is in a second state. The fluiddirection system also optionally provides for incubating the first andthe second molecules together for a specific period of time.

In some embodiments, the voltage regulator system of the a system ordevice in accordance with the invention applies a pulsed electric fieldcomprising a first state in which an electric field produces a specificvoltage or electric current in the microchannel for a first specificcontrollable period of time followed by a second state in which theelectric field is not applied for a second specific controllable periodof time. Alternatively, in the first state a first electric field isapplied that produces a first voltage or first current at a first levelin the microchannel for a first period of time, and in the second statea second electric field is applied that produces a second voltage orsecond current at a second level for a second period of time. Suchperiods of time are optionally determined based upon the rate ofinjection of the first or second molecule into the microchannel, or uponthe intermittent flow of the second molecule. Integrated systems ordevices in accordance with the invention optionally detect one or moreof fluorescence, chemiluminescence, or radiation from the molecules inthe microchannel.

Many additional aspects of the invention will be apparent upon completereview of this disclosure, including uses of devices and systems inaccordance with the invention, methods of manufacture of devices andsystems in accordance with the invention, and kits for practicingmethods in accordance with the invention. For example, kits comprisingembodiments of any devices or systems for performing one or more pulsedfield mobility shift assay in accordance with the invention, or elementsthereof, in conjunction with packaging materials (e.g., containers, orsealable plastic bags) and instructions for using the devices topractice the methods herein, are also contemplated.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1, panels A and B, schematically illustrate a microchannelcontaining areas of higher and lower fluorescence resulting from theincreased mobility of a labeled product.

FIG. 2 schematically represents an exemplary microfluidic device inaccordance with the invention that is capable of performing a pulsedfield assay.

FIG. 3 is a representation of fluorescein fluorescence intensity in apulsed electric field.

FIG. 4 is a representation of fluorescence intensity of 2 peptides in apulsed electric field.

FIG. 5 is a graph constructed from exemplary calculations used to findoptimal sensitivity parameters of a pulsed field mobility shift assay.

FIG. 6, panels A and B, schematically illustrate an exemplarymicrofluidic junction channel arrangement suitable for use with pulsedfield mobility shift assays. .

FIG. 7 is a schematic diagram of a channel arrangement suitable for usewith pulsed field mobility shift assays.

FIG. 8 shows representative data from a mobility shift assay in a highthroughput format in accordance with the invention.

FIG. 9 shows a portion of the data in FIG. 8 in more detail.

FIG. 10 compares dose response measurements produced by a pulsed fieldmobility shift assay with measurements produced by a standard mobilityshift assay.

FIG. 11 is a schematic view of an integrated system comprising amicrofluidic device incorporating an embodiment of a pulsed fieldmobility shift assay in accordance with the invention.

DETAILED DISCUSSION OF THE INVENTION

The methods and devices of the current invention increase the throughputof mobility shift assays carried out in microfluidic devices. Briefly,the invention provides devices and methods that can increase thethroughput rate of assays comprising the electrophoretic separation ofmaterials through the application of a pulsed electric field thatprovides specific pulses of voltage and/or electric current through amicrochannel in the microfluidic device.

Methods and devices in accordance with the current invention can be usedto perform pulsed field mobility shift assays for a variety ofapplications. For example, methods and devices in accordance with theinvention can be utilized in microfluidic devices to maximize throughputby decreasing the throughput time for a variety of assays. Embodimentsof the invention can significantly improve the throughput of processesinvolving the screening of large libraries such as combinatoriallibraries. Those screening processes can be time consuming due to theaggregation of time requirements for assay of each component in thelibrary. While microfluidic devices reduce the time required for suchlarge screening processes, the time required for assays that have lowthroughput or non-optimized throughput can also be significantlyimproved. Embodiments of the methods and devices of the currentinvention increase throughput by decreasing the amount of time needed toperform mobility shift assays in microfluidic devices.

To decrease the time needed to perform mobility shift assays on amicrofluidic device, embodiments of the current invention apply pulsedelectric fields through the microchannels of the microfluidic device.The manner in which these pulses are applied, which may be coordinatedwith the flow of one or more of the molecules being assayed, can beoptimized for a specific assay(s), compensating for the specificproperties (such as electrophoretic mobility) of the molecules beingassayed.

Embodiments of the present invention also optionally include componentsfor controlling fluid flow, generating and controlling pulsed electricfields, reconstituting dried or immobilized samples, controllingtemperature, detecting and quantifying molecules, and positioningcomponents or devices (e.g., robotic devices).

In many applications of microfluidic devices, different species aremixed together so that they can interact to form one or more product.Any or all of the reactant species or reaction products may havedifferent electrophoretic mobilities. For example, an enzyme andsubstrate may be mixed within a microfluidic device to produce aproduct, and the substrate, product and enzyme may all have differentelectrophoretic mobilities. When a given electric field is appliedacross a microfluidic channel by placing electrodes at the end of thechannel, a species within the channel will move at a velocity largelydetermined by their respective electrophoretic mobilities. Theelectrophoretic mobility of a species is a function of the charge of thespecies, the size of the species, and the fluid through which thespecies is moving. So, for example, if all other factors influencingelectrophoretic mobility are equal, highly charged molecules will have agreater attraction for an oppositely charged electrode than moremodestly charged molecules, making more highly charged molecules traveltoward an oppositely charged electrode with a higher velocity. Moleculeswith differing electrophoretic mobilities can be separated inmicrofluidic devices, thus making it possible to monitor andcharacterize interactions between various molecules.

In embodiments of the invention, detection equipment downstream from areaction site in the microfluidic device can determine the concentrationof reactants and products based, in part, on the differingelectrophoretic mobilities of these components. For example, if anenzyme and a substrate are mixed at the start of a microchannel, and theenzyme and substrate interact as the mixture flows down the channel, theamount of the product of the interaction appearing downstream from thereaction site as a function of time will depend on factors such as therate of the reaction, and whether non-rate limiting amounts of reactants(substrate and enzyme) are provided. In addition, the relativeconcentrations of product, enzyme, and substrate measured downstream ofthe reaction site as a function of time will depend on the relativeelectrophoretic mobilities of those three species. For example, if themobility of the product is substantially lower than the mobility of thesubstrate, then changes in product and substrate concentrations withinthe microchannel will appear at the detector at different times.

Embodiments of the invention are able to use differences inelectrophoretic mobility to determine reaction rates for many differentreactant concentrations in very short periods of time and in very smallvolumes of fluids. The ability to assess reaction kinetics while thereaction is occurring and while the components are flowing through amicrochannel past a detector greatly increases the rate at which suchreactions can be assessed. This facilitates accurate high-throughputdetermination of reaction kinetics, and has a variety of otherapplications in regard to applications such as drug screening, nucleicacid sequencing, and enzyme kinetics. Embodiments of the invention canbe used to examine reactions between two or more components thatchemically join (by forming a covalent or non-covalent association) toform a new component or complex. Other reactions that can be examined byembodiments of the invention include reactions in which a reactant istransformed into a product by means of an enzyme, a catalyst, orexposure to electromagnetic radiation. Embodiments of the invention canalso be used to examine the spontaneous degradation of a component. Inmany embodiments, a first component and a second component are mixedtogether in a channel of a microfluidic device, where the componentsreact to form a product. The product, along with the reactants, can becharacterized by pulsed field assays in accordance with the invention.

Mobility Shift Assays

Mobility shift assays, which separate species by means of theirdiffering electrophoretic mobilities, can be used to track and analyzevarious biochemical reactions. The use of mobility shift assays has beendescribed in the previously cited patent entitled “Apparatus and Methodsfor Correcting for Variable Velocity in Microfluidic Systems,” byKopf-Sill et al.

Electrophoretic separation processes are often described in terms offlux and velocity. The flux J of a species is equal to the product ofthe velocity V at which molecules of the species move through a solutionand the concentration C of the species in that solution. Flux istypically expressed in units of number of molecules/(cross sectionalarea·time) or mass/(cross sectional area·time)).

Within a system, such as microfluidic device, the principle ofconservation of mass dictates that the total flux of all species (interms of mass) must be conserved within the system. So, for example, ina three component system, which has a first reactant with a massconcentration (i.e., mass/volume) C, and a velocity V₁, a secondreactant with velocity V₂ and mass concentration C₂, and a product, withvelocity V_(p) and mass concentration C_(p), the requirement that fluxbe conserved within the system dictates that the mass flux at a firstpoint w in a microchannel must equal the mass flux at a second point zin the channel as long as no material has entered or left the channelbetween those two points. In equation form, the conservation of flux isexpressed asV_(1w)C_(1w)+V_(2w)C_(2w)+V_(pw)C_(pw)=V_(1z)C_(1z)+V_(2z)C_(2z)+V_(pz)C_(pz).An alternative notation is[V₁C₁+V₂C₂+V_(p)C_(p)]_(w)=[V₁C₁+V₂C₂+V_(p)C_(p)]_(z). A more generalnotation that allows for an arbitrarily number of species at the firstand second points in the channel is: $\begin{matrix}{{\sum\limits_{i = 1}^{m}{ViCi}} = {\sum\limits_{j = 1}^{n}{VjCj}}} & (1)\end{matrix}$where C_(i) is the mass concentration (not molar concentration) of thei^(th) species at the first point in the channel, m is the number ofspecies at the first point in the channel, C_(j) is the massconcentration (not molar concentration) of the j^(th) species at thesecond point in the channel, and n is the number of species at thesecond point in the channel. The different species and different numberof species occurring at the first and second points in the channel allowfor any reactions that may have taken place in the channel. Thus, thesum of the mass concentration times the velocity of each of the speciesbefore a reaction is equal to the sum of the mass concentration timesthe velocity of each of the species after a reaction. In the cases whenthe reaction yields no net change in the total number of molecules, themolar flux as well as the mass flux is conserved.

When a reaction occurs as fluid flows through a channel in amicrofluidic device, and the channel is subjected to an electric field,the various components of the fluid (i.e., the molecular species)typically travel along the length of the channel at different velocitiesto a downstream position. At one or more downstream positions known asdetection points, one or more of the species is detected, typically bymeans of a label.

At one or more the detection points, the velocities of one or morereactants (V_(r1), V_(r2), V_(r3), etc.) or products (V_(p1), V_(p2),V_(p3), etc.) in the channel are determined by detecting those one ormore reactants or products. The velocities of the reactants or productsthat were not detected can be determined using the conservation of fluxequation (equation (1)), or by using the equations that describe howthose undetected reactants or products move in an electric field. Whenan electric field is applied to a channel filled with a fluid, theelectric field causes each species in the fluid to move down the channelat a velocity V_(tot), which is equal to the vector sum of theelectroosmotic velocity of the fluid V_(eo) and the electrophoreticvelocity of the species V_(ep):V _(tot) =V _(eo) +V _(ep)=(μ_(eo)+μ_(ep))E   (2)In equation (2), μ_(eo) and μ_(ep) are the electroosmotic mobility ofthe buffer and the electrophoretic mobility of the dissolved species,respectively, and E is the applied electric field. The electrophoreticmobility μ_(ep) is a function of the charge-to-hydrodynamic radius ratioof the species. The hydrodynamic radius of a species depends on the sizeand shape of the species, and the viscosity of the fluid. Theelectroosmotic mobility is a function of the surface properties of thechannel, and the permittivity and viscosity of the fluid.

Although products of reactions typically change velocity as they aremade from, or by, reactants, the velocity change is often considered tobe instantaneous because the product reaches its terminal velocity inthe system in a very short period of time. Thus, discounting thevelocity changes due to the pulsed electric fields, the velocity of aproduct is essentially constant immediately following formation of theproduct. Where the velocity changes significantly over time, due to,e.g., a change in the applied pulsed current, or where a change fromsubstrate to product results in a slow acceleration (or deceleration) inthe system, an “instantaneous velocity” equal to the change in distancefor a selected time can be determined by graphing distance against timeand taking the derivative of the resulting function at a particularpoint in time. There are situations where the electric field strength ina channel varies for reasons other than a change in the electric fieldbeing applied to that channel. For example, the electric field strengthin a channel could change because of a change in the ionic strength ofthe fluid in the channel.

Tracking of biochemical reactions in a microfluidic device through amobility assay (and particularly through use of a pulsed field mobilityassay as described herein) allows for the determination ofconcentrations of the species involved (and their concentration changesover time), the rate of the reaction, and enzyme kinetics of thereaction.

Velocity typically refers to the distance a selected component, or othermolecular species travels (l) divided by the time (t) required for thetravel. In traditional mobility shift assays, the velocities of thevarious species are essentially constant as those species travel alongthe length of a microchannel under a constant electric field in anelectrokinetic system. However, as explained below, the presentinvention applies pulsed electric fields to a microchannel in amicrofluidic device. These pulsed fields create a situation where thevelocities of the species in the microchannel are not constant.

The detection of results for many biochemical assays in conventionalexperimental methodology, as well as in microfluidic devices, isoftentimes based upon the use of fluorogenic or chromogenic labels. Insuch assays the quantum efficiency of a labeling fluorescent moiety orthe amount of colored label (chromophore) changes as a result of areaction, thus allowing for detection and/or characterization of thatreaction. However, for certain classes of assays, no fluorogenic orchromogenic labels are available. Assays in which the fluorescence orcolor a label does not change upon reaction are known as non-fluorogenicand non chromogenic assays respectively.

However, essentially any analysis in which a reactant is converted to aproduct with a different mobility than the reactant can be analyzed inan appropriately configured microfluidic device through use of amobility shift assay (especially pulsed field assays as describedherein). Such mobility shift assays are velocity-based or velocitogenicassays. One class of reactions that can be analyzed using velocity-basedassays in accordance with the invention is enzymatic reactions. Aspecific class of enzymes is kinases, which are enzymes that recognizespecific polypeptide sequences and phosphorylate them. Phosphorylationchanges the charge, mass and structure of the polypeptide substrate, sothe electrophoretic mobilities of the non-phosphorylated andphosphorylated species are different. As a consequence of this change inmobility, the non-phosphorylated and phosphorylated species move atdifferent rates in an applied electric field (either a traditionalsteady field or a pulsed field as in the current invention). Therefore,accurate rate determination and quantification of the phosphorylationreaction can be determined through measurement of the velocities andconcentrations of the various reactants and products. In other words, adistinct label does not have to be applied to the products todistinguish them from the reactants. It will be appreciated thatreactions involving kinases are only one example of reactions that maybe analyzed by embodiments of the invention. In other words, whiletracking/characterization of kinase reactions through use of theinvention is one possible use, many other reactions (both enzymatic andnot) are also amenable to use with the current invention.

In conventional mobility shift assays an enzyme (such as a kinase) and afluorescently-labeled substrate are introduced into a reaction channelcontinuously by means of a steady vacuum applied to a portion of themicrofluidic device downstream of the reaction channel. After flowingthrough the reaction channel, the reaction products and any remainingreactants flow through a separation channel. Signals from fluorescentlylabeled species are detected at the end of the separation channel.Signals emanate from both any remaining substrate and product, since thefluorescent label on the substrate remains on the substrate when it isconverted to product. When a species that inhibits the reaction isintroduced into the reaction channel, the detected signals will reflectthe inhibitor's effect on the reaction between enzyme and substrate. Thechange in detected signals will be determined by the degree ofinhibition, and on the spatial separation of the substrate and productsignals caused by the differing electrophoretic mobilities of thosespecies. The magnitude of the change in the fluorescent signalsindicates the potency of the inhibitor (e.g., percent inhibition), andthe spatial separation between the substrate and product signals dependson the electric field strength and the transit time in the separationchannel. For a small difference in mobility, either the field strengthor the transit time has to increase to achieve acceptable resolution. Anincrease in transit time causes an increase in diffusion and dispersion,which in turn reduces the assay resolution.

It will be appreciated that the concepts described herein fornon-fluorogenic assays are equally applicable for non-fluorescentsystems in which the label is other than a fluorophore. So, for example,embodiments of the invention may be applied to systems usingcolorimetric labels, radioactive labels, mass labels (e.g., such asmight be detected by mass spectrometry), or electrochemical labels. Itshould also be noted, that the terms non-fluorogenic assay and mobilityshift assay (regardless of whether the assay involves a constant orpulsed field) are used interchangeably herein. Both of these terms applyto assays based upon the difference in electrophoretic mobility betweena product and a reactant. So, for example, both those terms would applyto embodiments of the invention involving a non-chromogenic assay (anassay in which the color or intensity of a label does not change uponreaction), and a non-radiogenic assay (an assay in which the radioactivecomponent of the label is not modified by the reaction). Therefore, forsimplicity herein, when fluorogenic assays and non-fluorogenic assaysare discussed herein, similar comparisons apply for assays involvingradio labels, chromophore labels, pH labels, ionic labels, or othercommon labels known to one of skill in the art.

Non-fluorogenic assays can be carried out in a microfluidic device inwhich electroosmotic flow is occurring by periodically injectingreaction mixture into a separation channel in the device within whichreactants and products are separated by electrophoresis due to changesin the electrophoretic mobility resulting from the reaction. This typeof assay is referred to as a non-continuous assay. Assays employing suchperiodic injections are described in “Complexity and performance ofon-chip biochemical assays” by A. R. Kopf-Sill, T. Nikiforov, L. Bousse,R. Nagel, & J. W. Parce in Proceedings of Micro-and NanofabricatedElectro-Optical Mechanical Systems for Biomedical and EnvironmentalApplications, SPIE, Vol. 2978, San Jose, Calif., February 1997, p.172-179. The length of each periodic injection is typically on the orderof from about 0.0001 to 10 minutes, typically about 0.001 to 1 minute,often about 0.1 seconds to 10 second.

Mobility shift assays can also be carried out in microfluidic devices inwhich electroosmotic flow is occurring by continuously injecting thereaction mixture into a channel. This type of assay is referred to as acontinuous assay. In many biochemical reactions, the electrophoreticmobility μ_(ep) of a reactant molecule changes as a result of thetransformation of that reactant into a product by the reaction (e.g. amoiety is added to or cleaved from the reactant during the reaction).Such a change in electrophoretic mobility μ_(ep), and therefore velocityV_(tot) (see equation (2)), allows for the detection of non-fluorogenicreactions in a continuous flow format.

Determination of concentration of a reaction or assay product C_(p)through a mobility shift assay in a channel of a microfluidic device isalso possible. To determine the concentration of a species, the speciesis labeled (e.g., with a fluorophore or chromophore) and flowed down amicrofluidic channel and past a signal detector. In an exemplaryembodiment, the labeled species is a labeled first reactant having avelocity V_(r). This labeled first reactant produces a signal, such as afluorescent signal, detectable by the detector. The labeled firstreactant is converted to a labeled reaction product, the product havinga velocity V_(p). In the typical case, V_(r) does not equal V_(p),meaning that the signals from the labeled first reactant and the labeledproduct will not be detected by the detector at the same time becausethe reactant and product were physically separated as they traveled downthe microfluidic channel because of their differing velocities.Accordingly, the two signals can be separately detected. The relativesizes of the signals produced by the reactant and the product provide anindication of the relative concentrations of those species. In someembodiments, the concentrations of reactant, as indicated by the size ofthe signal produced by reactant, in the presence and in the absence ofthe reaction of interest can be compared. An absence of reaction can becreated by, for example, not adding another reactant that is required toinitiate the reaction. The signal pattern (i.e., signal as a function oftime) produced in the absence of reaction can serve as a baseline towhich signals produced in the presence of reaction can be compared.

In many embodiments of non-fluorogenic assays, a labeled reactantmolecule is converted by a reaction to a labeled product molecule bytreating the labeled reactant molecule with any physical component orforce that brings about the conversion. Such components or forcesinclude light, heat, electrical charge, a polymerization agent, acatalyst, and a binding molecule. In some embodiments, the label moietyon the labeled reactant and labeled product are identical. Inalternative embodiments, the label on the labeled reactant is modifiedso that a different label is present on the labeled product. Even withsuch a modification, however, the output (e.g., light of a particularwavelength) of the label typically does not change in a non-fluorogenicassay. Of course, where the label does change, the mobility shift assayscan also be applied, as the velocity will typically concomitantlychange.

In a microfluidic device in which an electric field is applied along thelength of a microchannel, charged species such as analytes, solventmolecules, reactants and products move along the microchannel by meansof the electrokinetic forces of electroosmosis and electrophoresis. Thenet mobility of each species is determined by the vectorial sum of theelectroosmotic and electrophoretic mobilities, the latter of which is afunction of the hydrodynamic radius-to-charge ratio of each species. Aspreviously discussed, the hydrodynamic radius-to-charge is proportionalto the velocity in a flowing system. In various embodiments, the fluidflow in the system may result from the application of electrokineticforces or pressure forces. During a chemical or biological reaction suchas ligand-receptor binding or antibody-antigen binding, the reactants ingeneral have different electrophoretic mobilities than the products. Thedifferences in mobilities are exploited in non-fluorogenic assays inaccordance with the invention in which the ability to separately detectreactants and products is not dependent on the production or quenchingof fluorescence as a consequence of the reaction. Instead, the mobilitydifference is used to separate the “reactant hole”, the change in signalfrom the baseline that reflects the decrease in reactant concentrationcaused by the consumption of the labeled reactant in the reaction, fromthe “product peak”, the change in signal from the baseline reflectingthe increase in product concentration caused by production of labeledproduct in the reaction. The difference between the baseline and thesignal pattern produced as a result of the reaction of interest takingplace under continuous flow conditions provides a signature from whichquantitative information on the reaction kinetics can be extracted.

FIG. 1 illustrates the basic concept of a continuous flow mobility shiftassay by applying the concept to a binding reaction A+B→P, where A is afluorescently labeled reactant, B is an unlabeled reactant, and P is aproduct. In FIG. 1, the fluorescently-labeled reactant molecules aredenoted by circles, the unlabeled reactant molecules are denoted bysquares, and the reaction product molecules are denoted by solidtriangles. Note that the reaction product molecules formed by thebinding reaction will also be labeled since the product molecules willcomprise the label from the labeled reactant. In the exemplaryembodiment of FIG. 1, the binding reaction is fast and has a highassociation constant K_(a), where K_(a)=[P]/[A][B] (the bracketsdenoting concentrations). The labeled reactant molecules (circles) areintroduced into the main channel 100 at a constant concentration. Beforethe binding reaction occurs, the total concentration of fluorescentlylabeled molecules in the channel 100 will be equal to that constantconcentration of labeled reactant molecules. This before-reaction totalconcentration of fluorescently labeled molecules will provide a baselinesignal at the detector. In this embodiment, the labeled reactant isassumed to have a lower electrophoretic mobility than the product, sothe labeled reactant moves more slowly down the channel 100 than theproduct. As indicated by the arrows 150, the direction of flow inchannel 100 is from left to right.

To initiate the binding reaction, a short pulse or plug 160 of unlabeledreactant molecules (squares) is injected into the main channel 100 froma side channel (not shown). Panel A of FIG. 1 shows the situation at theinstant the plug 160 of unlabeled reactant molecules (squares) isinjected into the main channel 100. After the injection, the unlabeledreactant molecules (squares) will bind to the labeled reactant molecules(circles), converting the labeled reactant molecules to relatively fastmoving product molecules (triangles). For purposes of simplifying theexample, the binding reaction is considered to occur instantaneously.Panel B of FIG. 1 illustrates the situation some time after the bindingreaction has occurred. In the time after the reaction, the faster movingproduct molecules (triangles) have moved down the channel 100 fasterthan the labeled reactant molecules (circles), thus giving rise to aportion 170 of the fluid in the microchannel 100 containing a higherlocal total concentration of fluorescent species (i.e., the sum of thefluorescence from the baseline concentration of labeled reactants andthe additional fluorescence of labeled products). Accordingly, thisfluid portion 170 will produce a fluorescent signal higher than thebaseline signal. The portion 180 of the fluid in the microchannel wherethe binding reaction took place will have a lower concentration oflabeled reactant molecules (circles) due to the depletion of thosemolecules by the reaction. Accordingly, that portion 180 of the fluidwill have a lower total concentration of fluorescent species that willproduce a fluorescent signal lower than the baseline signal.

Quantitatively, it is important to recognize that the portion 170 of thefluid containing the product occupies a larger volume in the channelthan the depleted portion 180 of the fluid reactant zone due to thehigher velocity of the product. Consequently, the maximum increase inconcentration of product in the channel will be less than the maximumdecrease in concentration of the labeled reactant since both the totalincrease and decrease consist of the same number of product and reactantmolecules respectively. Interestingly, when the fluorescence seen by astationary detector, which the fluid in channel 100 flows past, isplotted against time, the widths of the peak (caused by the product) andvalley (caused by depletion) are the same because the spatially widerproduct peak, which has been increased by a factor equal to the ratio ofproduct velocity V_(p) to reactant velocity V_(r), moves past thedetector faster by the same factor of V_(p)/V_(r). If the decrease inmolecular concentration of the product C_(r) in fluid region 180 isknown, the velocities V_(r) and V_(p) are known, the molecularconcentration of the product C_(p) in fluid region 170 can be calculatedas C_(p)=C_(r) (V_(r)/V_(p)), since in the exemplary reaction of thisembodiment the number of molecules of reactant consumed by the reactionis equal to the number of molecules of product produced by the reaction.

When a label detector (e.g., a photomultiplier tube or a photo diode) isplaced downstream of the injection point, whether the plug of fastermoving product will be partially or totally separated from the slowermoving depletion hole when the plug and hole reach the detector willdepend on the distance between the injection and detection points, thewidth of the injection plug, and the relative velocities of the labeledreactant and product. In the case of partial separation, a plot of thedetector signal as a function of time will show a peak followed by aplateau region and a valley. The ratio of the magnitude of the peak tovalley is C_(p)/C_(r), which, by algebraic manipulation, is equal toV_(r)/V_(p). The plateau region is lower in fluorescence than thebackground level. The ratio of the magnitude of the plateau region tothe valley is 1−(C_(p)/C_(r)) or 1−(V_(r)/V_(p)). In the case of totalseparation, the signal shows a peak and a valley separated by thebaseline fluorescence level instead of the plateau region.

As in conventional capillary electrophoresis, the resolution of theseparation is directly proportional to the electric field strength andthe transit time in the separation channel. In some situations, however,the length of time needed to obtain adequate resolution can bedeleterious to throughput. For a small shift in mobility, either thefield strength or the transit time has to increase in order to achieveacceptable resolution. For example, if the reactant and product onlydiffer by a small amount in their mobility, a relatively long transittime may be required to achieve acceptable resolution. The transit timecan be increased by increasing the distance from where the reactionoccurred to the detector. An increase in transit time, however, causesan increase in diffusion and dispersion that reduce the gains inresolution. Additionally, in the analysis of fast off-rate bindingreactions, it is desired to measure the reaction as soon as possible(i.e., as soon after the reaction occurs as possible). Therefore, thelong transit time that may be needed to adequately separate thereactants and products would distort the results of the analysis of thereaction.

Pulsed Field Mobility Shift Assays

Mobility shift assays that involve the application of pulsed electricfields can separate species much more rapidly than can mobility shiftassays that involve the application of constant electric fields.Embodiments of the present invention involve the application of a pulsedelectric field that induces a temporal change in the electrophoreticvelocity of the species in a sample as a fluid comprising the sampleflows through a microchannel. In exemplary embodiments, the flow of thefluid may result from electroosmosis or the application of pressure(e.g., a positive pressure or a vacuum). A detector located in a portionof the microchannel subjected to a pulsed electric field can measure thetime-dependent perturbations of the concentrations of species, fromwhich perturbations of the velocities of species can be derived throughapplication of the principle of mass flux conservation. The temporalchange in concentration of species can also be related to theelectrophoretic mobilities of the species. Embodiments of the inventioncan determine the extent of a reaction that involves a fluorescentlylabeled reactant and a fluorescently labeled product that have differentelectrophoretic mobilities, even when the fluorescent labels on thereactant and product are identical. The pulsed field assays of theinvention have a wide range of applications, and are especiallyadvantageous when applied to high throughput processes. Embodiments ofthe invention may also include continuous flow assays, which in somecases have higher throughput than assays requiring the additional stepof sample injection. Since embodiments of the current invention takeless time than existing assays, application of the invention may helpreduce problems in existing assays that stem from the dispersion ofsample bands.

In embodiments of the invention, an electric field being applied to amicrochannel is pulsed from a first value E₁ to a second value E₂. Invarious embodiments, either the first or second value can comprise azero value, so the electric field is pulsed from “off” to “on” or viceversa. In other embodiments, E₁ and E₂ both have non-zero values, so theelectric field is pulsed from a non-zero value to another non-zerovalue. Such non-zero pulses may be separated by periods in which noelectric field is applied. In some embodiments, the pulsed electricfield results in a voltage profile comprising pulses of oppositepolarity that are symmetric about a zero value (e.g., E₁ produces anegative voltage while E₂ produces a positive voltage of the samemagnitude). If the time durations of the symmetric pulses of oppositepolarity are equal, then the fluid being subjected to the pulsed fieldwill have no net electrokinetic motion induced by the application of thepulsed field. In other embodiments, zero net electrokinetic motion isachieved by applying a pulsed electric field in such a way that thefield produces voltage pulses of opposite polarity where the product ofvoltage pulse duration and pulse magnitude for each of the oppositepolarity pulses is equal.

A variety of electric field pulse patterns may be applied in variousembodiments of the invention. For example, in some embodiments the timeperiods of the electric field pulses and the time periods between pulsesare not equal. In those embodiments a pulse may comprise a shorter orlonger length of time than the period between pulses. In otherembodiments, the time periods of the pulses and the pause between pulsesare of equal length. In still other embodiments, the time period ofpulses or pauses between pulses may vary between successive pulses orsets of pulses (e.g., pairs of symmetric pulses of opposite polarity).Similarly, the magnitude of pulses may vary between successive pulses orsets of pulses.

A variety of different relationships between the electric field pulsepatterns applied to a microchannel and the manner in which samples areintroduced into that microchannel are compatible with the invention. Forexample, the electric field may be pulsed at frequencies higher or lowerthan the sample injection rate. In other words, the number of electricfield pulses per unit time applied to a microchannel can exceed or beless than the number of injections of sample per unit time into thatmicrochannel. It is also possible to apply one pulse in electric fieldfor each injection of sample.

A schematic representation of a microfluidic device in accordance withthe invention is shown in FIG. 2. A microfluidic device is a device inwhich fluid flows that has a feature, such as a chamber, channel, orreservoir with a cross-sectional dimension (e.g., depth, width, length,or diameter) of about 0.1 μm to about 500 μm. Exemplary microfluidicdevices are described in U.S. Pat. No. 5,942,443 entitled “HighThroughput Screening Assay Systems in Microscale Fluidic Devices”, whichissued Aug. 24, 1999. The microfluidic device 202 in FIG. 2 comprises aseparation channel 210 in which species can be separated by means ofdifferences in their respective electrophoretic mobilities. A pulsedelectric field can be applied to the separation channel 210 by applyingvoltages to electrodes placed in reservoirs 206 and 208. The separationchannel 210 is intersected by at least one other microscale channel(e.g., channel 216) disposed within the body of the device. Suchintersecting channels are used to transport materials into or out of theseparation channel 210. So, in the embodiment of FIG. 2, reservoirs 204and 205 could contain the various reactants whose interactions are to beassayed. In other embodiments, only a sub-portion or sub-region of aseparation channel is subjected to a pulsed field.

Although the embodiment of FIG. 2 comprises a single separation channel210, other embodiments may have two or more additional separationchannels are disposed within the microfluidic device 202. For example,in a microfluidic device with multiple separation channels, parallelpulsed field mobility shift assays could be carried out in differentseparation channels so that the effects of a particular enzyme has on anumber of different substrates could be simultaneously evaluated. Insome embodiments, a single microfluidic device could include from about1 to about 100 or more separation channels specifically configured toperform pulsed field assays in accordance with the invention.

The materials transported into or out of separation channel 210, such asreactant, products, buffers or reagents, may also be transported out ofor into reservoirs fluidly connected to the separation channel 210 byother microscale channels. These reservoirs may contain the materialsrequired to carry out assays in accordance with the invention, as wellas to any other operations that are carried out on the microfluidicdevice 202. Examples of different reservoirs that could be employed inembodiments of the invention are a reservoir containing a dilutionbuffer to be added upstream from the source of a reagent to dilute thereagent, and a reservoir that functions as waste well to store samplesafter a reaction or assay has been completed. The removal of thecompleted samples provides space in the channels to load and incubateother samples. In this fashion, the devices of the invention can be usedin a high throughput manner. The high throughput can be achieved bycontinuously loading, processing, and unloading samples into and out ofthe microchannels of the device. Increased throughput, in fact, is oneof the major benefits of the current invention. Because reactionproducts need not be flowed for such a long period of time (as intraditional electrophoretic separation assays), more samples can beloaded in the same period of time.

In other embodiments, materials may be introduced into the microfluidicdevice 202 from sources outside the device, as opposed to sources suchas reservoirs within the device. Materials outside the device can betransported to the device by means of a “capillary element” or othersimilar pipettor element. The capillary element can be temporarily orpermanently coupled to a source of fluidic material. In the embodimentof FIG. 2, a capillary interfaces with the microfluidic device 202 atintersection 220. Capillary elements can transport materials from suchexternal sources as microwell plates, solid substrates comprisinglyophilized components, or reservoirs in a microfluidic device. The useof capillary elements is described in U.S. Pat. No. 5,880,071 entitled“Electropipettor and Compensation Means for Electrophoretic Bias” by J.Wallace Parce et al., which issued Mar. 9, 1999.

In embodiments of the present invention, a dilution buffer is typicallyadded into the separation channel upstream of an optional shunt channel,so that the increase in flow rate due to the addition of buffer materialdownstream of its entry point may be counteracted by the reduction inpressure due to the shunt channel. Reagent materials, on the other hand,are typically added downstream of an optional shunt channel so that theyare added after the downstream flow rate in the main channel has beenreduced so that smaller quantities of reagent are added.

In general, microfluidic devices are planar in structure and areconstructed from an aggregation of planar substrate layers whereinfeatures such as microchannels are formed at the interface of thevarious substrate layers. In some embodiments, the microchannels arefabricated by etching, embossing, mólding, ablating or otherwisefabricating into a surface of a first substrate grooves. A secondsubstrate layer is subsequently overlaid on the first substrate layerand bonded to it in order to cover the grooves in the first layer, thuscreating sealed features within the interior portion of the device.Microfluidic devices in accordance with the invention can take a varietyof forms, and do not need to have a layered planar structure. Forexample, microfluidic devices in accordance with the invention mayinclude aggregations of various components such as capillary tubes andindividual chambers that are pieced together to provide the integratedelements of the complete device.

Manufacturing of these microscale elements into the surface of thesubstrates can be carried out through any number of microfabricationtechniques that are well known in the art. For example, lithographictechniques are optionally employed in fabricating, e.g., glass, quartzor silicon substrates, using methods well known in the semiconductormanufacturing industries such as photolithographic etching, plasmaetching or wet chemical etching. Alternatively, micromachining methodssuch as laser drilling, micromilling and the like are optionallyemployed. Similarly, for polymeric substrates, well known manufacturingtechniques may also be used. These techniques include injection moldingor stamp molding methods wherein large numbers of substrates areoptionally produced using, e.g., rolling stamps to produce large sheetsof microscale substrates, or polymer microcasting techniques where thesubstrate is polymerized within a micromachined mold. Furthermore,various combinations of such techniques are optionally combined toproduce the microelements present in the current invention.

The substrates used to construct the microfluidic devices of theinvention are typically fabricated from any number of differentmaterials, depending upon such factors as the nature of the samples tobe assayed and the specific reactions and/or interactions being assayed.For some applications, the substrate can optionally comprise a solidnon-porous material. For example, the substrate layers can be composedof silica-based materials (such as glass, quartz, silicon, fused silica,or the like), polymeric materials or polymer coatings on materials (suchas polymethylmethacrylate, polycarbonate, polytetrafluoroethylene,polyvinylchloride, polydimethylsiloxane, polysulfone, polystyrene,polymethylpentene, polypropylene, polyethylene, polyvinylidine fluoride,acrylonitrile-butadiene-styrene copolymer, parylene or the like),ceramic materials, or metallic materials (e.g., wherein the metalmaterials are coated with an electrical insulation layer such as a metaloxide).

The surface of a substrate layer may be of the same material as thenon-surface areas of the substrate or, alternatively, the surface maycomprise a coating on the substrate base. For example, a surface may becoated in order to reduce or prevent electroosmotic flow in theseparation channel. Furthermore, if the surface is coated, the coatingoptionally can cover either the entire substrate base or can coverselect portions of the substrate base (e.g., only the separation channelis so coated). For example, in the case of glass substrates, the surfaceof the glass of the base substrate may be treated to provide surfaceproperties that are compatible and/or beneficial to one or more sampleor reagent being used. Such treatments include derivatization of theglass surface, e.g., through silanization or the like, or throughcoating of the surface using, e.g., a thin layer of other material suchas a polymeric or metallic material. Derivatization using silanechemistry is well known to those of skill in the art and can be readilyemployed to add, e.g., amine, aldehyde, or other functional groups tothe surface of the glass substrate, depending upon the desired surfaceproperties. Further, in the case of metal substrates, metals that arenot easily corroded under potentially high salt conditions, appliedelectric fields, and the like are optionally preferred.

One major advantage of the methods and devices of the present inventionis that pulsed field assays enhance the throughput rate through theseparation channel (and hence, the throughput through the microfluidicdevice). A detector (e.g., to read the fluorescence levels indicatingelectrophoretic separation of species) can thus be located much closerto the start of the separation channel instead of near the end (e.g.,compare detector 260 and detector 240 in FIG. 2). With the methods anddevices of the present invention, the detector no longer has to waituntil the species are electrophoretically separated in the regularsteady state field. Instead, as in FIG. 2, the detector (again, e.g.,detector 240) can be right near the beginning of the separation channel.

In an exemplary application of the device 202 in FIG. 2 to a pulsedfield assay in accordance with the invention, channel 210 is fluidlycoupled to channel region 212, which is connected to capillary element220 that can access samples stored outside of the device in a microwellplate or the like. For example, capillary element 220 can access amicrowell plate (or even numerous microwell plates accessed via arobotic armature) that contains a number of compounds to be screenedwithin the microfluidic device. Within the reaction channel region, 212,reactants such as an enzyme and a putative substrate, or a receptor anda putative ligand, interact.

The fluidic material (or more typically, a mixture of fluidic materials)drawn through capillary element 220 can be mixed with a buffer in orderto, e.g., dilute the sample to a proper concentration for the necessaryassays/reactions to occur and/or to help dilute unwanted sample storagematerials such as DMSO. To accomplish such, a quantity of buffer can beflowed from a buffer reservoir (not shown) and mixed with the contentsof the main channel. Alternatively, additional fluidic materials inplace of, or in addition to, buffers are optionally flowed into thechannel from, e.g., other reservoir(s).

In the embodiment of FIG. 2, the fluidic material drawn through thecapillary is joined by materials flowing from reservoirs 204 and 205. Insome embodiments, the material drawn through the capillary is a compoundbeing screened as a potential inhibitor to a reaction between thematerials introduced from reservoirs 204 and 205. Accordingly, thematerial from the two reservoirs could be enzymes, substrates,receptors, ligands, nucleic acids, antibodies, antigens, or otherreaction system constituents amenable to be assayed through anon-fluorogenic pulsed field mobility shift assay. The mixture of thereactants from the capillary and the reservoirs is flowed into reactionchannel region 212. In reaction channel region 212 the fluidic materialsundergo their putative interaction. Reaction channel region 212therefore has the proper conditions for such reaction to take place,such as the proper temperature, pH, and osmolality.

Flow of materials through reaction region 212 can be by any type orcombination of types of flow such as electrokinetic or pressure based.In many embodiments pressure based flow is used.

The reaction components next travel into separation channel region 210.Voltage pulses between an electrode in well/reservoir 206 and one inreservoir 208 in FIG. 2, drive the reactants, molecular species throughthe separation channel region. As explained previously, pressure drivenflow can be used in the main separation channel in conjunction with theelectrokinetic flow resulting from the pulsed electric field to movematerials along and also to help optimize sensitivity of the pulsedfield assay.

The pulsed electric fields used to electrophoretically separateconstituents in channel region 210 can be optionally modified induration (e.g., the time on and the time off can be modified, see,above) as well as in intensity (e.g., the voltage level is optionallycontrollable, see, above). It should be noted that detector 240 isplaced at the beginning of the separation channel region in FIG. 2(i.e., close to the reaction region). This again illustrates a majoradvantage of the current methods and devices. Because of the pulsedfields of the present invention, the detector can be placed closer tothe reaction channel region instead of at the end of the separationchannel region (e.g., as with detector 260 in FIG. 2).

The above example, illustrates that the methods and devices of thecurrent invention (i.e., pulsed field electrophoretic separation ofdifferent components) are easily adaptable to many differentexperimental situations and can be adapted to many different uses whichmay be ancillary and/or additional to other uses of the device.

The pulsing of the voltage (e.g., turning on and off of the voltage)applied to the electrodes immersed in reservoirs 206 and 208 creates apulsed electric field through the separation microchannel 210 in FIG. 2.The pulsed field will cause a fluorescently (or otherwise labeled)reactant and/or product to have an oscillatory fluorescent signal atdetector 240 with the same frequency as the applied pulsed field. Thiswill be true if the labeled molecules in question areelectrophoretically active and/or if the microchannel supportselectroosmotic flow.

Such pulsing is done at a quicker rate than the rate of intermittentinjections of, e.g., substrate into the main reaction channel (i.e.,injection from well 205 into reaction channel 212). In otherembodiments, different ratios of pulse to injection will apply. In somecontexts, e.g., if the system being assayed is very stable and theinterval between sample injection and arrival of the peak of the sampleconcentration at a detector is known, then optionally only one pulse persample injection would be needed. In embodiments wherein the pulse isfrom zero to a set value (e.g., as opposed to situations where the pulsegoes from one non-zero value to another non-zero value), an optimumlength of time during which the field is on (i.e. pulsed) willoptionally exist. However, in embodiments wherein the system is notentirely predictable, or in situations wherein it is desirous to collectmore data, e.g., to improve signal/noise, more than one pulse isoptionally applied for each sample injection. Optionally, as pulsedurations become shorter, the amplitude-change in the signal during thepulses gets small, thus setting a guideline to the upper frequency ofpulsing in some embodiments.

In some embodiments, the intermittent injection of a sample is done fora period of time that is at least as long as one complete pulse of theelectric field/voltage. In situations where two or more samples (e.g.,samples of an enzyme and samples of a substrate) are injectedintermittently, the interjections typically overlap each other eitherpartially or completely. Of course, in this non-limiting example,instead of substrate, a reaction enhancer, a reaction inhibitor, or acompetitive substrate could be the molecule being intermittentlyinjected into the channel 212. In various embodiments, the values of Ecan range from less than 10 V/cm to up to about 1,000 V/cm or more, fromless than 100 V/cm to up to about 500 V/cm or more, from less than 250V/cm to up to about 300 V/cm or more.

A simple example of the application of a pulsed electric field to achannel in a microfluidic device is illustrated in FIG. 3. FIG. 3 showsa measured level of fluorescence from fluorescein (anelectrophoretically active molecule) continuously flowed via a pressuredriven flow in a pulsed electric field (with electroosmosis suppressedin the microchannel). The electric field is pulsed between an “on” (i.e.a non-zero electric field is applied) and an “off” (i.e. no electricfield is applied) state. As can be seen in FIG. 3, when the pulsed fieldis ‘on’ (i.e., points 1, 2, and 3), the intensity of fluoresceinfluorescence (and hence its concentration) is lower than when the pulsedfield is ‘off’ (i.e., points 4, 5, and 6) where the level offluorescence is higher.

The results in FIG. 3 can be explained by the previously discussedconservation of flux equations. Since the amount of fluorescein flowingthrough the channel remains the same, regardless of whether the pulsedfield is “on” or “off”, the mass flux, J, of the fluorescein will beconstant. In the absence of an electric field, i.e., when the pulsedfield is off, the velocity of the fluorescein in FIG. 3 flowing in themicrochannel is V_(p), which the velocity resulting from the applicationof the pressure. When the electric field is applied, however, thefluorescein will have an additional velocity component fromelectrophoresis V_(ep). Accordingly, the conservation of mass fluxequation dictates that the concentration of the fluorescein at thedetection point when the electric field is on, C_(on), will be relatedto the concentration of the fluorescein at the detection point when theelectric field is off, C_(off), by the relationJ=C _(on)(V _(p) +V _(ep))=C _(off) V _(p).   (3)Solving for C_(on) gives the relationC_(on)=C_(off)(V_(p)/(V_(p)+V_(ep))). When, as in this embodiment, thevelocities produced by the pressure and electrophoretic forces are inthe same direction, the velocity V_(p) will be less than the velocityV_(p)+V_(ep). Thus, as is shown in FIG. 3, the measured value of C_(on)will be lower than the measured value of C_(off) by a factor ofV_(p)/(V_(p)+V_(ep)).

Electroosmotic flow was suppressed in the embodiment of FIG. 3. Ingeneral, however, when an electric field is applied to a channel, whichoccurs during the “on” portion of a pulsed field comprising “on” and“off” states, the velocity of species in the channel will have twoadditional velocity components: one from electrophoresis (V_(ep)) andone from electroosmosis (V_(eo)). Therefore, on a time scale shortenough so that there is no significant change in the composition of thefluid due to chemical reactions, the mass flux of a species across across section of the separation channel when the pulsed electric fieldis off (J₀) and when the pulsed field is on (J₁) must be conserved.Since the principle of conservation of mass flux dictates that J₀ mustequal J₁, the concentrations and velocities of the species in the on andoff states are related byJ ₀ =C ₀ V ₀ =J ₁ =C ₁ V ₁,   (4)where C₁ is the concentration of species in when the pulsed field is on,and C₀ is the concentration of the species when the pulsed field is off.Expressing the velocities in the two states in terms of their componentvelocities gives, $\begin{matrix}{\frac{C_{0}}{C_{1}} = {\frac{V_{1}}{V_{0}} = {\frac{\left( {V_{p} + V_{ep} + V_{eo}} \right)}{V_{p}}.}}} & (5)\end{matrix}$When the electric field is off, the pressure driving force produces theonly velocity component. When the electric field is on there areadditional electrophoretic and electroosmotic velocity components.

It is useful to define a net electrokinetic velocity V_(ek), which isequal to the sum of the electrophoretic and electroosmotic velocitycomponents (V_(ep)+V_(eo)). If the net electrokinetic velocity V_(ek) isin same direction as pressure driven velocity V_(p), then C₁ will alwaysbe lower than C₀. This was the case in the embodiment of FIG. 3. In FIG.3, when the pulsed field was on (i.e., in regions 1, 2, and 3) theconcentration C₁ was lower than the concentration C₀ when the field wasoff (i.e., regions/times 4, 5, and 6).

Additionally, both electrokinetic velocities (i.e., V_(ep) and V_(eo))are directly proportional to the applied electric field, E, by theirrespective mobilities μ_(ep) and μ_(eo). Thus, if we define a netelectrokinetic mobility μ_(ek), which is equal to (μ_(ep)+μ_(eo)), weobtain the relationV_(ek)=μ_(ek)E.   (6)Inserting this expression for V_(ek) into equation (5) and solving forμ_(ek) gives the relation $\begin{matrix}{\mu_{ek} = {{V_{p}\left( \frac{\frac{C_{0}}{C_{1}} - 1}{E} \right)}.}} & (7)\end{matrix}$It should be noted that in the absence of electroosmosis (e.g., as whenthe wall of a microchannel is specifically coated to preventelectroosmosis, see, below), μ_(ek) reduces to the electrophoreticmobility of the species μ_(ep).

While FIG. 3 illustrates the concentration and velocity changes of asingle species in a pulsed electric field, pulsed electric fieldmobility shift assays are typically used to track changes inconcentration and velocity of multiple species in a microchannel. Thesemultiple species could be, for example, the species involved in a kinasereaction or a binding reaction. Therefore two species or more areusually assayed. The results from an embodiment where both a fluorescentreactant and a fluorescent product are electrophoretically active isshown in FIG. 4. In FIG. 4, two peptides with different electrophoreticmobilities were drawn into a separation channel and flowed under thepulsed field methods of the present invention. In the separation channelof this embodiment, electroosmosis was suppressed. The pulsed electricfield comprised an on state in which an electric field was applied tothe channel, and an off state when no electric field was applied. Therate of the pulsed field, i.e., how quickly the electric field waspulsed on and off, was one second on and one second off. The firstpeptide comprised a polypeptide with six amino acids units and a netcharge of −2. Region 1 of FIG. 4 shows the detected fluorescence whenonly this peptide was present in the separation channel. Next, a nineamino acid polypeptide with a net −2 charge was flowed into theseparation channel for 30 seconds. The resulting fluorescence is shownin region 2 of FIG. 4. Finally, the separation channel was again filledwith the original six amino acid peptide. The resulting fluorescence isshown in region 3 of FIG. 4. The difference in fluorescence intensitiesbetween the six amino acid peptide and the nine amino acid peptideresults from the difference in the electrophoretic mobilities of thosemolecules. Thus, the results shown in FIG. 4 indicate that changes inthe electrophoretic mobility of a molecule, as might occur when themolecule is transformed in a reaction or binds to another molecule, canbe detected by methods in accordance with the invention.

An additional benefit of using a mobility shift assay employing a pulsedelectric field, as opposed to a convention mobility shift assayemploying a constant electric field, is that the pulsed field assay canbetter analyze binding reactions that have a fast “off-rate”. In a fastoff-rate binding reaction, the binding reaction between the species israpid and reversible. If the time required to perform a conventionalmobility shift assay is long enough, the continuous application of therequired electric field might separate the bound and unbound species.Separating those species would affect the reaction equilibrium,distorting the results of the assay. This could occur, for example, whenassaying a binding reaction between a receptor and a ligand. Forinstance, for a binding reaction with an equilibrium association bindingconstant (K_(eq)) of 10⁶ M and an on-rate (k_(on)) of 10⁷ to 10⁸ M⁻¹s⁻¹,the off rate is on the order of 1 to 10 s⁻¹. In such a case, a 1-secondpulse should not perturb the equilibrium significantly, while a20-second separation time, which is commonly required for traditionalmobility shift assays, would. Moreover, the signal in the pulsed fieldmethod is self-calibrating since the concentration ratio, (C₀/C₁), willnormalize out effects such as pipetting error and fluorescence quenchingdue to reaction.

Additionally, the methods/devices of the present invention areoptionally calibrated/modified in order to optimize the sensitivity ofthe pulsed field assay. To illustrate the optimization of a pulsed fieldassay, a pulsed field assay involving two detectable species, species aand species b, will be optimized. In various embodiments, the twospecies could be a similarly labeled reactant and product respectively.In order to optimize the exemplary pulsed field assay, the value ofΔC₁/C₀ is maximized, wherein ΔC₁ equals the difference in concentrationof the two species when the pulsed field is on and C₀ equals theconcentration of one of the species when the pulsed field is off.Similarly, in embodiments where the pulsed electric field alternatesbetween two non-zero electric fields, ΔC₁ would be the difference inconcentration between the species when one of the two non-zero electricfields is applied, while C₀ would be the concentration of one of thespecies when the other field is applied. To maximize ΔC₁/C₀, themagnitude of the electric field E in the on state, the pressure inducedvelocity V_(p), or the electrokinetic mobilities of the two species(μ_(a) and μ_(b)) can be manipulated.

For example, if two species are put through the exemplary pulsed fieldassay, the conservation of mass flux equation for species ‘a’ would be$\begin{matrix}{{\frac{C_{1a}}{C_{0a}} = \frac{V_{p}}{V_{p} + {\mu_{a}E}}},} & (8)\end{matrix}$while the corresponding equation for species ‘b’ would be$\begin{matrix}{\frac{C_{1b}}{C_{0b}} = {\frac{V_{p}}{V_{p} + {\mu_{b}E}}.}} & (9)\end{matrix}$The overall sensitivity of the process can be expressed as$\begin{matrix}{\frac{\Delta\quad C_{1}}{C_{0}} = {{\frac{C_{1a}}{C_{0a}} - \frac{C_{1b}}{C_{0b}}} = {{V_{p}\left( {\frac{1}{V_{p} + {\mu_{a}E}} - \frac{1}{V_{p} + {\mu_{b}E}}} \right)}.}}} & (10)\end{matrix}$Equation (10) can be rearranged to give $\begin{matrix}{\frac{\Delta\quad C_{1}}{C_{0}} = {\frac{E}{V_{p}}{\frac{\left( {\mu_{b} - \mu_{a}} \right)}{\left( {1 + \frac{\mu_{a}E}{V_{p}}} \right)\left( {1 + \frac{\mu_{b}E}{V_{p}}} \right)}.}}} & (11)\end{matrix}$Therefore, the parameters V_(p), E and (μ_(b)-μ_(a)) can be manipulatedto maximize ΔC₁/C₀, thus maximizing the overall sensitivity of thepulsed field assay. Equation (11) can be rewritten as $\begin{matrix}{\frac{\Delta\quad C_{1}}{C_{0}} = {\left( {\mu_{b} - \mu_{a}} \right)E\quad{{V_{p}\left( \frac{1}{\left( {V_{p} + {\mu_{a}E}} \right)\left( {V_{p} + {\mu_{b}E}} \right)} \right)}.}}} & (12)\end{matrix}$Differentiating ΔC₁/C₀, with respect to the experimentally controllablevariable V_(p) (i.e., the component of species velocity produced bypressure) gives $\begin{matrix}{\frac{\partial\left( \frac{\Delta\quad C_{1}}{C_{0}} \right)}{\partial V_{p}} = {\left( {\mu_{b} - \mu_{a}} \right)E\left\{ {\frac{1}{\left( {V_{p} + {\mu_{a}E}} \right)\left( {V_{p} + {\mu_{b}E}} \right)} + \frac{- V_{p}}{\left( {V_{p} + {\mu_{b}E}} \right)\left( {V_{p} + {\mu_{a}E}} \right)^{2}} + \frac{- V_{p}}{\left( {V_{p} + {\mu_{a}E}} \right)\left( {V_{p} + {\mu_{b}E}} \right)^{2}}} \right\}}} & (13)\end{matrix}$To maximize ΔC₁/C₀ with respect to V_(p), we set $\begin{matrix}{\frac{\partial\left( \frac{\Delta\quad C_{1}}{C_{0}} \right)}{\partial V_{p}} = 0.} & (14)\end{matrix}$Combining equations (13) and (14), and simplifying the result gives$\begin{matrix}{{1 - \frac{V_{p}}{V_{p} + {\mu_{a}E}} - \frac{V_{p}}{V_{p} + {\mu_{b}E}}} = 0.} & (15)\end{matrix}$Solving equation (15) for V_(p) gives the value of V_(p) at which theassay sensitivity is at its maximum:V _(p)=√{square root over (μ_(a)μ_(b))}E   (16)

If in the embodiment of FIG. 4, the electrokinetic mobility of species‘a’ were equal to 1.28×10⁻⁴ cm²/vs and the electrokinetic mobility ofspecies ‘b’ (μ_(b)) were equal to 1.16×10⁻⁴ cm²/vs, then ΔC₁/C₀ ismaximized when the pressure flow (V_(p)) is equal to 0.156 cm/s for apulsed field E of 1278 V/cm. This is shown in FIG. 5, which shows ΔC₁/C₀as a function of V_(p) for that embodiment. FIG. 5 shows there exists arelatively stable plateau region around the point of maximumsensitivity, which means that for that embodiment there is a wide rangeof pressure driven flow conditions that provide near optimum assaysensitivity.

Optimization of Channel Geometry

Although the channel configuration in the microfluidic device of FIG. 2is suitable for carrying out embodiments of the invention, the channelconfigurations can be further optimized to improve the results obtainedfrom embodiments of the invention. Since embodiments of the inventionreduce the time required to perform the separation step of a mobilityshift assay, the sample being assayed will not have to flow as far downthe separation channel 210 as it would have to in a standard (i.e.,constant electric field) mobility shift assay. Accordingly, a detector,such as the detector 240 in FIG. 2, can be placed closer to the channeljunction where the sample is introduced into the separation channel 210than a detector 260 suitable for use with a standard mobility shiftassay. In the embodiment of FIG. 2, the channel junction where thesample is introduced into the separation channel 210 is at theintersection of channels 212, 216, and 210. The quality of signalresulting from the pulsed field assay depends upon the fielddistribution near the channel junction. For example, if a sample plugexperiences a non-uniform electric field across the cross-section of amicrochannel, then the velocity of the charged molecules will bedifferent at different locations across the channel. Hence the signalpeak (from the particles) will spread out in time domain and the signalquality will be degraded.

FIG. 6 a shows a channel junction similar to the channel junction inFIG. 2 between channels 212, 210, and 216. The electric field beingapplied to the separation channel 210 results from a voltage appliedbetween electrodes in reservoirs 206 and 208 in FIG. 2. The 90° turnmade by the electric field as it turns from channel 216 into channel 210produces a very non-uniform electric field in the vicinity of thechannel junction. The non-uniform electric field is higher near corner601, and lower on the opposite wall. The non-uniformity seen in thechannel junction of FIG. 6 a can be reduced by applying the electricfield by placing electrodes in reservoirs disposed on opposite sides ofthe separation channel 210. This could be accomplished by placingelectrodes in reservoirs 206 and 207 in FIG. 2, and by fluidlyconnecting reservoir 207 with separation channel by means of a channel217 (not shown in FIG. 2). FIG. 6 b shows a channel junction thatinterfaces with two electrode-containing reservoirs through channels 216and 217. The channel junction in FIG. 6 b enhances the uniformity of theelectric field across the channel junction, thus improving the resultsfrom a pulsed field mobility shift assay. The embodiment of FIG. 6 bcould be further optimized, however, because the sharp corners at thechannel junction still may cause localized high electric field regions.

The angle and width of the two electrode channels 216 and 217 can bevaried to further optimize the uniformity of the electric field at thechannel junction. FIG. 7 shows one junction geometry thus optimized bymeans of a numerical simulation of the electric field lines. By varyingthe side channel geometry and angle, optimized design features for anelectrode junction are optionally achieved with a near uniform electricfield. The channels leading to electrodes 216, 217 are tilted at anangle of 135°, thus minimizing the high electric field at the corners.Additionally, the width of the reaction channel 212 (59 μm) is less thanthat the width of the separation channel 210 (74 μm). By creating a nearuniform electric field at the channel junction, the results from pulsedfield mobility shift assays in accordance with the invention will beimproved.

Exemplary Screening Applications

The methods and devices of the present invention can be used to maximizethe throughput of microfluidic devices in which serially introducedsamples are assayed. The “throughput” of a microfluidic device istypically defined as the number of different materials that can beserially processed by the device per unit time. Materials that arescreened at rates greater than 1 material per minute within a singlechannel of a microfluidic device are generally termed high throughput,while screening of compounds at a rate greater than 1 compound per 10seconds generally falls into the ultra-high throughput category.Decreasing the amount of time required to perform separations in amicrochannel allows the spacing between serially introduced materials tobe reduced, thus allowing a greater number of different materials to beserially introduced into the microfluidic device per unit time. Thecloser that samples are capable of being loaded, the more samples thatcan be analyzed per unit time.

Spacers and/or buffers are optionally used to keep different reactionsseparated and/or prevent mixing of samples. For example, a buffer isoptionally loaded into a channel after each plug of sample in order toseparate samples form one another and prevent contamination betweensamples. Alternatively, while an enzyme solution may be flowedcontinuously in a separation channel, different putative substrates maybe flowed into the separation channel in plugs. Such plugs are typicallyseparated by materials such as buffers. A sample plug includes aninitial sample aliquot and any products produced by incubation orreaction of the initial sample aliquot. The buffer plugs optionally cancomprise immiscible fluids to decrease diffusion. Buffer plug lengthsare calculated in the same way as sample plug lengths based onconsiderations such as diffusivity and/or dispersion of the material.For example a buffer plug is typically 500 μm to 5 mm, preferably 600 μmto 3 mm or 850 μm to 1 mm. The last buffer plug loaded or added into achannel or the device is optionally longer, e.g., 500 μm to about 10 mm,to allow for flow pinching.

In an exemplary embodiment, the pulsed field mobility shift assay wasused for screening for Protein Kinase A (PKA) inhibitors. Samples weredrawn from a 96 well plate into a microfluidic device through acapillary. The plate contained 7 different concentration samples (0.15μM, 0.32 μM, 0.63 μM, 1.54 μM, 2.52 μM, 5.05 μM, and 30.75 μM) of H9 atrandomly chosen wells. Except for a single well containing the 30.75 μMconcentration of H9, two wells contained each of the other sixconcentrations of H9. The last column of each row of the well platecontained a marker used to indicate the end of row. In this embodiment,the marker was 1 μM of 100% converted PKA product. Once in the reactionchannel of the microfluidic device, the pressure driven velocity of thesample was set at 0.028 cm/s and the applied electric field was set to997 V/cm. FIG. 8 shows the raw data collected for the entire 96 wellplate. Each sample was sipped for 30 seconds separated by a 30 secondbuffer sip. The data from row F in FIG. 8 are shown in more detail inFIG. 9. The data in FIG. 9 clearly shows the change in amplitude of thesignal when H9 at the 0.32 and 2.52 μM concentration were sipped fromrow F of the plate. From the data in FIGS. 8 and 9, the parameter C₁/C₀,which is the ratio of the fluorescence measured while the electric fieldis on to the fluorescence measured while the field is off, wasdetermined for each of the seven H9 concentrations. That parameter wasalso measured in the absence of H9 to provide a baseline value. Thedeviation from the baseline valueΔC/C=((C₁/C₀)|_(H9Sample)−(C₁/C₀)|_(buffer)), was evaluated for each H9sample. The experimental measurements were then converted to percentinhibition by dividing the ΔC/C value for a concentration of H9 by theΔC/C value corresponding to 100% percent inhibition. These percentinhibition values were used to generate the dose response curve in FIG.10. The dose response data from the pulsed electric field mobility shiftassay (diamonds) were compared to dose response data obtained from astandard (i.e., constant electric field) mobility shift assay (squares).The data from the two methods show remarkable agreement and predict sameK_(i) value for the inhibitor. The K_(i) value was found to be 3.5 μM.These results clearly show that the pulse field technique can be used toperform high throughput mobility based assays.

Integrated Systems, Methods and Microfluidic Devices of the Invention

The microfluidic devices of the invention can include numerous optionalvariant embodiments including methods and devices for, e.g., fluidtransport, temperature control, detection and the like.

Although the devices and systems specifically illustrated herein aregenerally described in terms of the performance of a few operations, orof one particular operation (e.g., pulsed field assays), it will bereadily appreciated from this disclosure that the flexibility of thesesystems permits easy integration of additional operations into thesedevices. For example, the devices and systems described will optionallyinclude structures, reagents and systems for performing virtually anynumber of operations both upstream and downstream from the operationsspecifically described herein (e.g., upstream and/or downstream ofpulsed field separation of fluidic materials as described herein, etc.).Such upstream operations include such operations as sample handling andpreparation, e.g., extraction, purification, amplification, cellularactivation, labeling reactions, dilution, aliquotting, and the like.Similarly, downstream operations optionally include similar operations,including, e.g., further separation of sample components, labeling ofcomponents, assays and detection operations, electrokinetic orpressure-based injection of components or the like.

The microfluidic devices of the present invention can include otherfeatures of microscale systems, such as fluid transport systems whichdirect particle/fluid movement within and to the microfluidic devices,as well as the flow of fluids to and through various channels orregions, etc. Various combinations of fluid flow mechanisms can beutilized in embodiments of the present invention (e.g., pressure drivenflow through the reaction and separation channel regions in FIG. 2).Additionally, various types of fluid flow mechanisms can be utilized inseparate areas of microfluidic devices of the invention. For example,the pulsed electrokinetic flow optionally occurs within the separationchannel region (210 in FIG. 2) while, e.g., pressure driven flowoptionally (and additionally) occurs throughout reaction channel 212 andseparation channel 210. Flow of fluidic components such as reagents(e.g., as in to and from the main separation channel), etc., canincorporate any movement mechanism set forth herein (e.g., fluidpressure sources for modulating fluid pressure inmicrochannels/microreservoirs/etc.; electrokinetic controllers formodulating voltage or current in microchannels/micro-reservoirs/etc.;gravity flow modulators; magnetic control elements for modulating amagnetic field within the microfluidic device; use of hydrostatic,capillary, or wicking forces; or combinations thereof, etc.).

The microfluidic devices of the invention can also include fluidmanipulation elements such as parallel stream fluidic converters, i.e.,converters which facilitate conversion of at least one serial stream ofreagents into parallel streams of reagents for parallel delivery to areaction site or reaction sites within the device, e.g., as wherein thedevice has multiple pulse field separation channels or regions. Thesystems herein optionally include mechanisms such as valve manifolds anda plurality of solenoid valves to control flow switching, e.g., betweenchannels and/or to control pressure/vacuum levels in the, e.g.,microchannels. Additionally, molecules, etc. are optionally loaded intoone or more channels of a microfluidic device through one sippercapillary fluidly coupled to each of one or more channels and to asample or particle source, such as a microwell plate.

In the present invention, materials such as proteins, antibodies,enzymes, substrates, buffers, or the like are optionally monitoredand/or detected, e.g., the presence of a component of interest can bedetected, an activity of a compound can be determined, separation offluidic materials can be monitored or an effect of a modulator, e.g., onan enzyme's activity, can be measured. Depending upon the detectedsignal measurements, decisions are optionally made regarding subsequentfluidic operations, e.g., whether to assay a particular component indetail to determine, e.g., kinetic information or, e.g., whether, when,or to what extent to shunt a portion of a fluidic material from a mainchannel into a second channel (e.g., flowing a fluidic material into asecond channel once it has been separated from a mixture of fluidicmaterials). For example, prior to testing putative substrates against anenzyme in the pulsed field assay, the substrates may bemonitored/sorted, etc. and only certain ones directed to be testedagainst certain enzymes (i.e., in the pulsed field assay).

In brief, the systems described herein optionally include microfluidicdevices, as described above, in conjunction with additionalinstrumentation for controlling fluid transport, flow rate and directionwithin the devices, detection instrumentation for detecting or sensingresults of the operations performed by the system, processors, e.g.,computers, for instructing the controlling instrumentation in accordancewith preprogrammed instructions, receiving data from the detectioninstrumentation, and for analyzing, storing and interpreting the data,and providing the data and interpretations in a readily accessiblereporting format. For example, the systems herein optionally include avalve manifold and a plurality of solenoid valves to control flowswitching between channels and/or to control pressure/vacuum levels inthe channels.

Temperature Control

Various embodiments of the present invention can control temperatures toinfluence numerous parameters or reaction conditions, e.g., those inthermocycling reactions (e.g., PCR, LCR). Additionally, the presentinvention can control temperatures in order to manipulate reagentproperties, etc. In general, and in optional embodiments of theinvention, various heating methods can be used to provide a controlledtemperature in the involved miniaturized fluidic systems. Such heatingmethods include both joule and non-joule heating.

Non-joule heating methods can be internal, i.e., integrated into thestructure of the microfluidic device, or external, i.e., separate fromthe microfluidic device. Non-joule heat sources can include, e.g.,photon beams, fluid jets, liquid jets, lasers, electromagnetic fields,gas jets, electron beams, thermoelectric heaters, water baths, furnaces,resistive thin films, resistive heating coils, peltier heaters, or othermaterials, which provide heat to the fluidic system in a conductivemanner. Such conductive heating elements transfer thermal energy from,e.g., a resistive element in the heating element to the microfluidicsystem by way of conduction. Thermal energy provided to the microfluidicsystem overall, increases the temperature of the microfluidic system toa desired temperature. Accordingly, the fluid temperature and thetemperature of the molecules within, e.g., the microchannels of thesystem, are also increased in temperature. An internal controller in theheating element or within the microfluidic device optionally can be usedto regulate the temperature involved. These examples are not limitingand numerous other energy sources can be utilized to raise, control, ormodulate, the fluid temperature in the microfluidic device.

Non-joule heating units can attach directly to an external portion of achip of the microfluidic device. Alternatively, non-joule heating unitscan be integrated into the structure of the microfluidic device. Ineither case, the non-joule heating is optionally applied to onlyselected portions of chips in microfluidic devices (e.g., such asreaction areas, detection areas, etc.) or optionally heats the entirechip of the microfluidic device and provides a uniform temperaturedistribution throughout the chip. For example, a non-joule heatingmethod optionally only heats the reaction channel area (e.g., region 212in FIG. 2) so that the correct reaction parameters are set.

A variety of methods can be used to lower fluid temperature in themicrofluidic system, through use of energy sinks. Such an energy sinkcan be a thermal sink or a chemical sink and can be flood, time-varying,spatially varying, or continuous. A thermal sink can include, amongothers, a fluid jet, a liquid jet, a gas jet, a cryogenic fluid, asuper-cooled liquid, a thermoelectric cooling means, e.g., peltierdevice or an electromagnetic field.

In general, electric current passing through the fluid in a channelproduces heat by dissipating energy through the electrical resistance ofthe fluid. Power dissipates as the current passes through the fluid andgoes into the fluid as energy as a function of time to heat the fluid.The following mathematical expression generally describes a relationshipbetween power, electrical current, and fluid resistance: wherePOWER=power dissipated in fluid: I=electric current passing throughfluid; and R=electric resistance of fluid.POWER=I²R

The above equation provides a relationship between power dissipated(“POWER”) to current (“I”) and resistance (“R”). In some of theembodiments of the invention, wherein electric current is directedtoward moving a fluid (where such is utilized, e.g., in the separationchannel region 210 in FIG. 2 where the pulsed fields are used toelectrophoretically separate species), a portion of the power goes intokinetic energy of moving the fluid through the channel. Joule heatinguses a selected portion of the power to heat the fluid in the channel ora selected channel region(s) of the microfluidic device and can utilizein-channel electrodes. See, e.g., U.S. Pat. No. 5,965,410, which isincorporated herein by reference in its entirety for all purposes. Sucha channel region is sometimes narrower or smaller in cross section thanother channel regions in the channel structure. The small cross sectionprovides higher resistance in the fluid, which increases the temperatureof the fluid as electric current passes therethrough. Alternatively, theelectric current can be increased along the length of the channel byincreased voltage, which also increases the amount of power dissipatedinto the fluid to correspondingly increase fluid temperature.

Joule heating permits the precise regional control of temperature and/orheating within separate microfluidic elements of the device of theinvention, e.g., within one or several separate channels, withoutheating other regions where such heating is, e.g., unnecessary orundesirable. For example, joule heating is optionally used within, e.g.,a microchannel leading from a well holding a putative substrate toensure proper temperature for folding, etc., but not within the reactionchannel region (e.g., 212 in FIG. 2) or vice versa depending upon thespecific reaction conditions. Because the microfluidic elements involvedare extremely small in comparison to the mass of the entire microfluidicdevice in which they are fabricated, such heat remains substantiallylocalized, e.g., it dissipates into and from the device before itaffects other fluidic elements. In other words, the relatively massivedevice functions as a heat sink for the separate fluidic elementscontained therein.

To selectively control the temperature of fluid or material of a regionof, e.g., a microchannel, the joule heating power supply of theinvention can apply voltage and/or current in several optional ways. Forinstance, the power supply optionally applies direct current (i.e., DC),which passes through one region of a microchannel and into anotherregion of the same microchannel that is smaller in cross section inorder to heat fluid and material in the second region. This directcurrent can be selectively adjusted in magnitude to complement anyvoltage or electric field applied between the regions to move materialsin and out of the respective regions.

In order to heat the material within a region, without adverselyaffecting the movement of a material, alternating current (i.e., AC) canbe selectively applied by a power supply. The AC used to heat the fluidcan be selectively adjusted to complement any voltage or electric fieldapplied between regions in order to move fluid into and out of variousregions of the device (including the pulsed electric fields used inseparation of species). Thus, in options wherein AC is used within theinvention, the AC is optionally used in non-pulse field assay areas(e.g., only in reaction areas such as region 210 in FIG. 2, etc.). Insome nontypical embodiments, AC is optionally used within any area ofthe devices of the invention, including within pulsed field separationareas such as region 210 in FIG. 2. However, in the nontypicalembodiments wherein AC is used within pulsed field separation regions,the AC field is optionally applied at a much higher frequency than thevoltage pulses used for separation of molecules. Thus the AC highfrequency response can be filtered out from the low frequency pulsedfield signal of interest. Alternating current, voltage, and/or frequencycan be adjusted, for example, to heat a fluid without substantiallymoving the fluid. Alternatively, the power supply can apply a pulse orimpulse of current and/or voltage, which will pass through onemicrochannel region and into another microchannel region to heat thefluid in the region at a given instance in time. This pulse can beselectively adjusted to complement any voltage or electric field appliedbetween the regions in order to move materials, e.g., fluids or othermaterials, into and out of the various regions. Pulse width, shape,and/or intensity can be adjusted, for example, to heat a fluidsubstantially without moving the fluid or any materials within thefluid, or to heat the material(s) while moving the fluid or materials.Still further, the power supply optionally applies any combination ofDC, AC, and pulse, depending upon the application. The microchannel(s)itself optionally has a desired cross section (e.g., diameter, width ordepth) that enhances the heating effects of the current passed throughit and the thermal transfer of energy from the current to the fluid(e.g., in addition to, or alternative to, any cross-sectional geometryused to manipulate dispersion rate and/or average velocity of fluidicmaterials). Again, such above described joule heating is optionally usedonly in select areas of the present invention and can be entirelyseparate from, or integrated with the electric pulses used in the pulsedfield assays.

Because electrical energy is optionally used to control temperaturedirectly within the fluids contained in the microfluidic devices, themethods and devices of the invention are optionally utilized inmicrofluidic systems that employ electrokinetic material transportsystems, as noted herein. Specifically, the same electrical controllers,power supplies and electrodes can be readily used to control temperaturecontemporaneously with their control of material transport. See, infra.In some embodiments of the invention, the device provides multipletemperature zones by use of zone heating. On such example apparatus isdescribed in Kopp, M. et al. (1998) “Chemical amplification:continuous-flow PCR on a chip” Science 280(5366): 1046-1048.

As can be seen from the above, the elements of the current invention canbe configured in many different arrangements depending upon the specificneeds of the molecules, etc. under consideration and the parameters ofthe specific assays/reactions involved. Again, the above non-limitingillustrations are only examples of the many differentconfigurations/embodiments of the invention.

Fluid Flow

A variety of controlling instrumentation and methodology is optionallyutilized in conjunction with the microfluidic devices described herein,for controlling the transport and direction of fluidic materials and/ormaterials within the devices of the present invention by, e.g.,pressure-based or electrokinetic control, etc.

In the present system, the fluid direction system controls thetransport, flow and/or movement of samples, other reagents, etc. intoand through the microfluidic device. For example, the fluid directionsystem optionally directs the movement of one or more fluid (e.g.,samples, buffers) etc. into, e.g., the reaction channel regions, theseparation channel regions, etc. The fluid direction system alsooptionally directs the simultaneous or sequential movement of fluidicmaterials into one or more channels, etc., e.g., in situations whereinmore than one separation channel exits, etc. Additionally, the fluiddirection system can optionally direct the shunting of portions offluidic materials into shunt microchannels and the like.

The fluid direction system also optionally iteratively repeats the fluiddirection movements to help create high throughput screening, e.g., ofthousands of samples. Alternatively, the fluid direction systemoptionally repeats the fluid direction movements to a lesser degree ofiterations to create a lower throughput screening (applied, e.g., whenthe specific analysis under observation requires, e.g., a longincubation time when a higher throughput format would be counterproductive) or the fluid direction system utilizes a format of highthroughput and low throughput screening depending on the specificrequirements of the assay, e.g., ancillary actions upstream and/ordownstream of the pulsed field assays of the invention, may involvelower throughput activity. Additionally, the devices of the inventionoptionally use a multiplex format to help achieve high throughputscreening, e.g., through use of a series of multiplexed sipper devicesor multiplexed system of channels coupled to a single controller forscreening in order to increase the amount of samples analyzed in a givenperiod of time. Again, the fluid direction system of the inventionoptionally controls the flow (timing, rate, etc.) of samples, reagents,buffers, etc. involved in the various optional multiplex embodiments ofthe invention.

One method of achieving transport or movement of particles throughmicrofluidic devices is by electrokinetic material transport. Ingeneral, electrokinetic material transport and direction systems includethose systems that rely upon the electrophoretic mobility of chargedspecies within an electric field applied to the structure. Such systemsare more particularly referred to as electrophoretic material transportsystems. In the current invention, electrokinetic transport is used asthe method of fluid transport when species are electrophoreticallyseparated in the pulsed field mobility shift assays (i.e., in theseparation channel region) and optionally within other areas of thedevices as well, in order to move reagents, etc. to and from variouslocations of the device. For example, in some embodiments,electrokinetic flow is also applied in, e.g., the separation channelregion (see, e.g., FIG. 2). Such electrokinetic flow can be a steadyfield (or flow) onto which, or on top of which, the pulsed field isapplied.

Electrokinetic material transport systems, as used herein, and asoptional aspects of the present invention, include systems thattransport and direct materials within a structure containing, e.g.,microchannels, microreservoirs, etc., through the application ofelectrical fields to the materials, thereby causing material movementthrough and among the areas of the microfluidic devices, e.g., cationswill move toward a negative electrode, while anions will move toward apositive electrode. Movement of fluids toward or away from a cathode oranode can cause movement of particles suspended within the fluid (oreven particles over which the fluid flows). Similarly, the particles canbe charged, in which case they will move toward an oppositely chargedelectrode (indeed, it is possible to achieve fluid flow in one directionwhile achieving particle flow in the opposite direction). In someembodiments of the present invention, the fluid and/or particles, etc.within the fluid, can be immobile or flowing.

For optional electrophoretic applications of the present invention, thewalls of interior channels of the electrokinetic transport system areoptionally charged or uncharged. For example, as detailed previously,some separation channel regions are specifically coated to preventelectroosmotic flow within the region. Typical electrokinetic transportsystems are made of glass, charged polymers, and uncharged polymers. Theinterior channels are optionally coated with a material which alters thesurface charge of the channel. A variety of electrokinetic controllersare described, e.g., in Ramsey WO 96/04547, Parce et al. WO 98/46438 andDubrow et al., WO 98/49548 (all of which are incorporated herein byreference in their entirety for all purposes), as well as in a varietyof other references noted herein.

To provide appropriate electric fields, the system of the currentmicrofluidic device optionally includes a voltage controller that iscapable of applying selectable voltage levels, simultaneously, to, e.g.,each of the various microchannels and micro-reservoirs. Such a voltagecontroller is optionally implemented using multiple voltage dividers andmultiple relays to obtain the selectable voltage levels. Alternatively,multiple independent voltage sources are used. For example, differentindependent voltage sources, etc. are optionally used for the separationchannel region where pulsed field assays occur and for all other areasof the device where electrokinetic flow is used for material transport(as opposed to separation). The voltage controller is electricallyconnected to each of the device's fluid conduits via an electrodepositioned or fabricated within each of, or a subset of, the pluralityof fluid conduits (e.g., microchannels, microreservoirs, etc.). In oneembodiment, multiple electrodes are positioned to provide for switchingof the electric field direction in the, e.g., microchannel(s), therebycausing the analytes to travel a longer distance than the physicallength of the microchannel. Use of electrokinetic transport to controlmaterial movement in interconnected channel structures was described in,e.g., WO 96/94547 to Ramsey. An exemplary controller is described inU.S. Pat. No. 5,800,690. Modulating voltages are concomitantly appliedto the various fluid areas of the device to affect a desired fluid flowcharacteristic, e.g., continuous or discontinuous (e.g., a regularlypulsed field causing the sample to oscillate its direction of travel)flow of labeled components toward a waste reservoir. Particularly,modulation of the voltages applied at the various areas can move anddirect fluid flow through the interconnected channel structure of thedevice.

The controlling instrumentation discussed above is also optionally usedto provide for electrokinetic injection or withdrawal of fluidicmaterial downstream of a region of interest to control an upstream flowrate. The same instrumentation and techniques described above are alsoutilized to inject a fluid into a downstream port to function as a flowcontrol element.

The current invention also optionally includes other methods of fluidtransport, e.g., available for situations in which electrokineticmethods are not desirable. See, above. For example, fluid transport anddirection, etc. are optionally carried out, in part, in a pressure-basedsystem to, e.g., avoid electrokinetic biasing during sample mixing,e.g., in the reaction channel region before species are separated.Additionally, as described above, pressure based flow or other similarflow such as wicking, etc. is used within the separation channel regionas a controllable variable to help optimize the pulsed fieldelectrophoretic separations of the invention. See, above. Highthroughput systems typically use pressure induced sample introduction.Pressure based flow is also desirable in systems in which electrokinetictransport is also used. For example, as detailed throughout the currentinvention, pressure based flow is optionally used for introducing andreacting reagents in a system in which the products areelectrophoretically separated. In the present invention molecules areoptionally loaded and other reagents are flowed through themicrochannels or microreservoirs, etc. using, e.g., electrokinetic fluidcontrol and/or under pressure.

Pressure is optionally applied to the microscale elements of theinvention, e.g., to a microchannel, microreservoir, region, etc. toachieve fluid movement using any of a variety of techniques. Fluid flowand flow of materials suspended or solubilized within the fluid,including cells or molecules, is optionally regulated by pressure basedmechanisms such as those based upon fluid displacement, e.g., using apiston, pressure diaphragm, vacuum pump, probe or the like to displaceliquid and raise or lower the pressure at a site in the microfluidicsystem. The pressure is optionally pneumatic, e.g., a pressurized gas,or uses hydraulic forces, e.g., pressurized liquid, or alternatively,uses a positive displacement mechanism, e.g., a plunger fitted into amaterial reservoir, for forcing material through a channel or otherconduit, or is a combination of such forces. Internal sources includemicrofabricated pumps, e.g., diaphragm pumps, thermal pumps, lamb wavepumps and the like that have been described in the art. See, e.g., U.S.Pat. Nos. 5,271,724; 5,277,566; and 5,375,979 and Published PCTApplication Nos. WO 94/05414 and WO 97/02347.

In some embodiments, a pressure source is applied to a reservoir or wellat one end of a microchannel to force a fluidic material through thechannel. Optionally, the pressure can be applied to multiple ports atchannel termini, or, a single pressure source can be used at a mainchannel terminus. Optionally, the pressure source is a vacuum sourceapplied at the downstream terminus of the main channel (e.g., well 211in FIG. 2 which optionally has a vacuum source attached to it to drawsamples through the microchannels of the invention) or at the termini ofmultiple channels. Pressure or vacuum sources are optionally suppliedexternally to the device or system, e.g., external vacuum or pressurepumps sealably fitted to the inlet or outlet of channels or to thesurface openings of micro-reservoirs, or they are internal to thedevice, e.g., microfabricated pumps integrated into the device andoperably linked to channels or they are both external and internal tothe device. Examples of microfabricated pumps have been widely describedin the art. See, e.g., published International Application No. WO97/02357.

These applied pressures, or vacuums, generate pressure differentialsacross the lengths of channels to drive fluid flow through them. In theinterconnected channel networks described herein, differential flowrates or volumes are optionally accomplished by applying differentpressures or vacuums at multiple ports, or, by applying a single vacuumat a common waste port and configuring the various channels withappropriate resistance to yield desired flow rates. As discussed above,this is optionally done with multiple sources or by connecting a singlesource to a valve manifold comprising multiple electronically controlledvalves, e.g., solenoid valves.

Hydrostatic, wicking and capillary forces are also optionally used toprovide fluid flow of materials such as reagents, buffers, etc. in theinvention. See, e.g., “Method And Apparatus For Continuous Liquid FlowIn Microscale Channels Using Pressure Injection, Wicking AndElectrokinetic Injection,” by Alajoki et al., U.S. Pat. No. 6,416,642.In using wicking/capillary methods, an adsorbent material or branchedcapillary structure is placed in fluidic contact with a region wherepressure is applied, thereby causing fluid to move towards the adsorbentmaterial or branched capillary structure. Furthermore, the capillaryforces are optionally used in conjunction with, e.g., electrokinetic orpressure-based flow in the channels, etc. of the present invention inorder to pull fluidic material, etc. through the channels. Additionally,a wick is optionally added to draw fluid through a porous matrix fixedin a microscale channel or capillary.

Use of a hydrostatic pressure differential is another optional way tocontrol flow rates through the channels, etc. of the present invention.For example, in a simple passive aspect, an enzyme suspension isdeposited in a reservoir or well at one end of a channel at sufficientvolume or height so that the enzyme suspension creates a hydrostaticpressure differential along the length of the channel by virtue of,e.g., the enzyme suspension reservoir having greater height than a wellat an opposite terminus of the channel. Typically, the reservoir volumeis quite large in comparison to the volume or flow-through rate of thechannel, e.g., 10 microliter reservoirs or larger as compared to a 100micrometer channel cross section.

The present invention optionally includes mechanisms for reducingadsorption of materials during fluid-based flow, e.g., as are describedin “Prevention Of Surface Adsorption In Microchannels By Application OfElectric Current During Pressure-Induced Flow”, U.S. Pat. No. 6,458,259by Parce et al. In brief, adsorption of components, proteins, enzymes,markers and other materials to channel walls or other microscalecomponents during pressure-based flow can be reduced by applying anelectric field such as an alternating current to the material duringflow. Alternatively, flow rate changes due to adsorption are detectedand the flow rate is adjusted by a change in pressure or voltage. Suchmechanisms are optionally used in areas of the device to, e.g.,transport reagents to the reaction channel region (e.g., 212 in FIG. 2)and are optionally not used within separation channel regions (e.g., 210in FIG. 2). In some embodiments, AC field flow, etc. are optionally usedin the separation regions of the invention (e.g., 210 in FIG. 2),however, at a much higher frequency than the pulsed voltages used forthe pulsed field assays, thus, the high frequency AC could be filteredout from the lower frequency pulses (i.e., the pulsed signals ofinterest in the pulsed field assays).

The invention also optionally includes mechanisms for focusing labelingreagents, enzymes, modulators, and other components into the center ofmicroscale flow paths, which is useful in increasing assay throughput byregularizing flow velocity, e.g., in pressure based flow, e.g., as aredescribed in “Focusing Of Microparticles In Microfluidic Systems”, U.S.Pat. No. 6,506,609 by H. Garrett Wada et al. In brief, sample materialsare focused into the center of a channel by forcing fluid flow fromopposing side channels into the main channel, or by other fluidmanipulation. Hence, e.g., at the detection regions in FIG. 2, thefluorescent species are focused in the microchannel allowing for bettermeasurements.

In an alternate embodiment, microfluidic systems of the invention can beincorporated into centrifuge rotor devices, which are spun in acentrifuge. Fluids and particles travel through the device due togravitational and centripetal/centrifugal pressure forces. Such forces,of course, would be used in conjunction with the electrokinetic forcesused in the pulsed field separation assays herein.

Fluid flow or particle flow in the present devices and methods isoptionally achieved using any one or more of the above techniques, aloneor in combination. For example, electrokinetic transport can be used inone area or region of a microfluidic device in order to, e.g., movematerial through a pulsed field assay region. Additionally, pressurebased flow could be used in a different region/area of the samemicrofluidic device where various fluidic materials (again, e.g.,enzymes or the like) are to be transported to a reaction region, etc.Myriad combinations of fluid transport methods can be combined invarious embodiments of the present invention depending upon the specificneeds of the system/assay being used.

Typically, the controller systems involved are appropriately configuredto receive or interface with a microfluidic device or system element asdescribed herein. For example, the controller, optionally includes astage upon which the device of the invention is mounted to facilitateappropriate interfacing between the controller and the device.Typically, the stage includes an appropriate mounting/alignmentstructural element, such as a nesting well, alignment pins and/or holes,asymmetric edge structures (to facilitate proper device alignment), andthe like. Many such configurations are described in the references citedherein.

Detection

In general, detection systems in microfluidic devices include, e.g.,optical sensors, temperature sensors, pressure sensors, pH sensors,conductivity sensors, and the like. Each of these types of sensors isreadily incorporated into the microfluidic systems described herein. Inthese systems, such detectors are placed either within or adjacent tothe microfluidic device or one or more microchannels, microchambers,microreservoirs or conduits of the device, such that the detector iswithin sensory communication with the device, channel, reservoir, orchamber, etc. For example, optical detectors (such as fluorescencedetectors) are placed in the detector regions 240 and/or 260 in FIG. 2.Detection systems can be used to, e.g., discern and/or monitor specificreactions, assays, etc. occurring within the microfluidic device, oralternatively, or additionally, to track, e.g., electrophoreticseparation of reaction components, etc. in the pulsed field assays. Thephrase “proximal,” to a particular element or region, as used herein,generally refers to the placement of the detector in a position suchthat the detector is capable of detecting the property of themicrofluidic device, a portion of the microfluidic device, or thecontents of a portion of the microfluidic device, for which thatdetector was intended. For example, a pH sensor placed in sensorycommunication with a microscale channel is capable of determining the pHof a fluid disposed in that channel. Similarly, a temperature sensorplaced in sensory communication with the body of a microfluidic deviceis capable of determining the temperature of the device itself.

Many different molecular/reaction characteristics can be detected inmicrofluidic devices of the current invention. For example, variousembodiments can detect such things as fluorescence or emitted light,changes in the thermal parameters (e.g., heat capacity, etc.) involvedin the assays, etc.

Examples of detection systems in the current invention can include,e.g., optical detection systems for detecting an optical property of amaterial within, e.g., the microchannels of the microfluidic devicesthat are incorporated into the microfluidic systems described herein.Such optical detection systems are typically placed adjacent to amicroscale channel of a microfluidic device, and optionally are insensory communication with the channel via an optical detection windowor zone that is disposed across the channel or chamber of the device.Again, such a detector is optionally placed at detector 240 in FIG. 2 tomeasure, e.g., fluorescence from reaction products in the separationchannel being separated in a pulsed field assay of the invention.

Optical detection systems of the invention include, e.g., systems thatare capable of measuring the light emitted from material within thechannel, the transmissivity or absorbance of the material, as well asthe material's spectral characteristics, e.g., fluorescence,chemiluminescence, etc. Detectors optionally detect a labeled compound,such as fluorographic, colorimetric and radioactive components. Types ofdetectors optionally include spectrophotometers, photodiodes, avalanchephotodiodes, microscopes, scintillation counters, cameras, diode arrays,imaging systems, photomultiplier tubes, CCD arrays, scanning detectors,galvo-scanners, film and the like, as well as combinations thereof.Proteins, antibodies, or other components that emit a detectable signalcan be flowed past the detector, or alternatively, the detector can moverelative to an array to determine molecule position (or, the detectorcan simultaneously monitor a number of spatial positions correspondingto channel regions, e.g., as in a CCD array). For example, a detector(or detectors) could track along separation channel 210 in FIG. 2 asvarious fluorescent components are pulsed through the channel. Examplesof suitable detectors are widely available from a variety of commercialsources known to persons of skill. See, also, The Photonics Design andApplication Handbook, books 1, 2, 3 and 4, published annually by LaurinPublishing Co., Berkshire Common, P.O. Box 1146, Pittsfield, Mass. forcommon sources for optical components.

As noted above, the present devices optionally include, as microfluidicdevices typically do, a detection window or zone at which a signal,e.g., fluorescence, is monitored. This detection window or zoneoptionally includes a transparent cover allowing visual or opticalobservation and detection of the assay results, e.g., observation of acolorimetric, fluorometric or radioactive response, or a change in thevelocity of calorimetric, fluorometric or radioactive component.

Another optional embodiment of the present invention involves use offluorescence correlation spectroscopy and/or confocal nanofluorimetrictechniques to detect fluorescence from the molecules in the microfluidicdevice. Such techniques are easily available (e.g., from Evotec,Hamburg, Germany) and involve detection of fluorescence from moleculesthat diffuse through the illuminated focus area of a confocal lens. Thelength of any photon burst observed will correspond to the time spent inthe confocal focus by the molecule. Various algorithms used for analysiscan be used to evaluate fluorescence signals from individual moleculesbased on changes in, e.g., brightness, fluorescence lifetime, spectralshift, FRET, quenching characteristics, etc.

The sensor or detection portion of the devices and methods of thepresent invention can optionally comprise a number of differentapparatuses. For example, fluorescence can be detected by, e.g., aphotomultiplier tube, a charge coupled device (CCD) (or a CCD camera), aphotodiode, or the like.

A photomultiplier tube is an optional aspect of the current invention.Photomultiplier tubes (PMTs) are devices which convert light (photons)into electronic signals. The detection of each photon by the PMT isamplified into a larger and more easily measurable pulse of electrons.PMTs are commonly used in many laboratory applications and settings andare well known to those in the art.

Another optional embodiment of the present invention comprises acharge-coupled device. CCD cameras can detect even very small amounts ofelectromagnetic energy (e.g., such that emitted by fluorophores in thepresent invention). CCD cameras are made from semi-conducting siliconwafers that release free electrons when light photons strike the wafers.The output of electrons is linearly directly proportional to the amountof photons that strike the wafer. This allows the correlation betweenthe image brightness and the actual brightness of the event observed.CCD cameras are very well suited for imaging of fluorescence emissionssince they can detect even extremely faint events, can work over a broadrange of spectrum, and can detect both very bright and very weak events.CCD cameras are well know to those in the art and several suitableexamples include those made by: Stratagene (La Jolla, Calif.),Alpha-Innotech (San Leandro, Calif.), and Apogee Instruments (Tucson,Ariz.) among others.

Yet another optional embodiment of the present invention comprises useof a photodiode to detect fluorescence from molecules in themicrofluidic device. Photodiodes absorb incident photons that causeelectrons in the photodiode to diffuse across a region in the diode thuscausing a measurable potential difference across the device. Thispotential can be measured and is directly related to the intensity ofthe incident light.

In some aspects, the detector measures an amount of light emitted fromthe material, such as a fluorescent or chemiluminescent material. Assuch, the detection system will typically include collection optics forgathering a light based signal transmitted through the detection windowor zone, and transmitting that signal to an appropriate light detector.Microscope objectives of varying power, field diameter, and focal lengthare readily utilized as at least a portion of this optical train. Thedetection system is typically coupled to a computer (described ingreater detail below), via an analog to digital or digital to analogconverter, for transmitting detected light data to the computer foranalysis, storage and data manipulation.

In the case of fluorescent materials such as labeled enzymes or productsor fluorescence indicator dyes or molecules, the detector optionallyincludes a light source that produces light at an appropriate wavelengthfor activating the fluorescent material, as well as optics for directingthe light source to the material contained in the channel. The lightsource can be any number of light sources that provides an appropriatewavelength, including lasers, laser diodes and LEDs. Other light sourcesare optionally utilized for other detection systems. For example,broadband light sources for light scattering/transmissivity detectionschemes, and the like. Typically, light selection parameters are wellknown to those of skill in the art.

The detector can exist as a separate unit, but is preferably integratedwith the controller system, into a single instrument. Integration ofthese functions into a single unit facilitates connection of theseinstruments with a computer (described below), by permitting the use offew or a single communication port(s) for transmitting informationbetween the controller, the detector and the computer. Integration ofthe detection system with a computer system typically includes softwarefor converting detector signal information into assay resultinformation, e.g., separation of species, concentration of a substrate,concentration of a product, presence of a compound of interest,interaction between various samples, or the like.

Computer

As noted above, any of the fluid direction system, the detection system,the electric pulse generator, etc. as well as other aspects of thecurrent invention described herein (e.g., temperature control, etc.),are optionally coupled to an appropriately programmed processor orcomputer that functions to instruct the operation of these instrumentsin accordance with preprogrammed or user input instructions, receivedata and information from these instruments, and interpret, manipulateand report this information to a user. As such, the computer istypically appropriately coupled to one or more of the appropriateinstruments (e.g., including an analog to digital or digital to analogconverter as needed).

The computer optionally includes appropriate software for receiving userinstructions, either in the form of user input into set parameterfields, e.g., in a GUI, or in the form of preprogrammed instructions,e.g., preprogrammed for a variety of different specific operations. Thesoftware then converts these instructions to appropriate language forinstructing the operation of, e.g., the fluid direction controller,electric pulse generator, etc. to carry out the desired operation.

For example, the computer is optionally used to direct a fluid directionsystem to control fluid flow, e.g., into and through a variety ofinterconnected microchannels (e.g., into and through the variousmicrochannels of the invention, such as the separation channelscomprising pulsed electric flow, etc.). Additionally, the fluiddirection system optionally directs fluid flow controlling which samplesare contacted with each other and/or with various reagents, buffers,etc. in, e.g., a reaction region or other region(s) in the microfluidicdevice. Furthermore, the fluid direction system optionally controls thecoordination of movements of multiple fluids/molecules/etc. concurrentlyas well as sequentially. For example, the computer optionally directsthe fluid direction system to direct the movement of at least a firstmember of a plurality of molecules into a first member of a plurality ofmicrochannels concurrent with directing the movement of at least asecond member of the plurality of molecules into one or more detectionchannel regions. For example, different samples can be separated indifferent pulsed field separation channels. Additionally oralternatively, the fluid direction system directs the movement of atleast a first member of the plurality of molecules into the plurality ofmicrochannels concurrent with incubating at least a second member of theplurality of molecules or directs movement of at least a first member ofthe plurality of molecules into the one or more detection or pulsedfield channel regions concurrent with incubating at least a secondmember of the plurality of molecules.

By coordinating channel switching, the computer controlled fluiddirection system directs the movement of at least one member of theplurality of molecules into the plurality of microchannels and/or onemember into a detection region at a desired time interval, e.g., greaterthan 1 minute, about every 60 seconds or less, about every 30 seconds orless, about every 10 seconds or less, about every 1.0 seconds or less,or about every 0.1 seconds or less. Each sample, with appropriatechannel switching as described above, remains in the plurality ofchannels for a desired period of time, e.g., between about 0.1 minutesor less and about 60 minutes or more. For example, samples optionallyremain in the reaction channels for a selected incubation time of, e.g.,2 minutes.

The computer then optionally receives the data from the one or moresensors/detectors included within the system, e.g., detector 240 in FIG.2, interprets the data, and either provides it in a user understoodformat, or uses that data to initiate further controller instructions,in accordance with the programming, e.g., such as in monitoring andcontrol of flow rates (e.g., as involved in separation of materialsthrough pulsed field analysis in the separation channel region),temperatures, applied voltages, pressures, and the like.

In the present invention, the computer typically includes software forthe monitoring and control of materials in the various microchannels,etc. For example, the software directs electric field pulses to controland direct flow as described above. Additionally the software isoptionally used to control electrokinetic, pressure-modulated, or thelike, injection or withdrawal of material. The computer also typicallyprovides instructions, e.g., to the controller or fluid direction systemfor switching flow between channels to help achieve a high throughputformat.

In addition, the computer optionally includes software for deconvolutionof the signal or signals from the detection system. For example, thedeconvolution distinguishes between two detectably different spectralelectrophoretic mobilities that were both detected, e.g., when labelintensity levels were measured in the separation channel.

Any controller or computer optionally includes a monitor, which is oftena cathode ray tube (“CRT”) display, a flat panel display (e.g., activematrix liquid crystal display, liquid crystal display), or the like.Data produced from the microfluidic device, e.g., fluorographicindication of selected molecules, is optionally displayed in electronicform on the monitor. Additionally, the data gathered from themicrofluidic device can be outputted in printed form. The data, whetherin printed form or electronic form (e.g., as displayed on a monitor),can be in various or multiple formats, e.g., curves, histograms, numericseries, tables, graphs and the like.

Computer circuitry is often placed in a box which includes, e.g.,numerous integrated circuit chips, such as a microprocessor, memory,interface circuits, etc. The box also optionally includes such things asa hard disk drive, a floppy disk drive, a high capacity removable drivesuch as a writeable CD-ROM, and other common peripheral elements.Inputting devices such as a keyboard or mouse optionally provide forinput from a user and for user selection of sequences to be compared orotherwise manipulated in the relevant computer system.

Exemplary Integrated System

FIG. 11 provides additional details regarding an exemplary integratedsystem that may be used with embodiments of the pulsed field assaydevices and methods of the invention. As shown, microfluidic device 202has main separation channel 210 disposed therein (e.g., a separationchannel wherein pulsed field assays occur). As depicted, the integratedsystem optionally includes pipettor channel 220 protruding frommicrofluidic device 202 for accessing a source of materials external tothe microfluidic system. In FIG. 11, the external source is a multiwellplate 708. A sample can be flowed from pipettor channel 220 by applyinga vacuum at reservoir 211 (or another point in the system), by applyingappropriate wicking arrangements, or by applying an electric field tothe pipettor channel to induce electrokinetic flow. Additionalmaterials, such as buffer solutions, substrate solutions, enzymesolutions, test molecules, fluorescence indicator dyes or molecules andthe like, can be flowed from reservoirs such as 204 or 205 and thenceinto a reaction channel and a separation channel.

Detector 706 is in sensory communication with a separation channel inthe microfluidic device 202 so that it can detect signals emanating fromlabeled materials flowing through the detection region. Detector 706 isoptionally coupled to any of the channels or regions of the device wheredetection is desired. Detector 706 is operably linked to computer 704,which digitizes, stores, and manipulates signal information detected bydetector 706.

Fluid direction system 702 controls voltage, pressure, etc. (or acombination of such), e.g., at the wells of the systems or through thechannels of the system, or at vacuum couplings fluidly coupled to mainseparation channel 210, or any other channel described above.Optionally, as depicted, computer 704 controls fluid direction system702. In one set of embodiments, computer 704 uses signal information toselect further parameters for the microfluidic system. For example, upondetecting the interaction between a particular sample and a firstreagent, the computer optionally directs addition of a second reagent ofinterest (e.g., a reaction inhibitor) into the system to be testedagainst that particular sample. In some embodiments, this same directionsystem controls the timing and strength of the pulsed electric fields inseparation channel 210 (e.g., through computer 704). In otherembodiments the pulsed fields are generated through a separate device,e.g., 720.

Temperature control system 710 controls joule and/or non-joule heatingat, e.g., the wells of the systems or through the channels of the systemas described herein. Optionally, as depicted, computer 704 controlstemperature control system 710. In one set of embodiments, computer 704uses signal information to select further parameters for themicrofluidic system. For example, upon detecting the desired temperaturein a sample in, e.g., reaction channel 212, the computer optionallydirects addition of, e.g., a potential binding molecule, etc. into thesystem to be tested against one or more samples.

Monitor 716 displays the data produced by the microfluidic device, e.g.,graphical representation of, e.g., separation or non-separation offluidic materials, interaction (if any) between samples, reagents, testmolecules, etc. Optionally, as depicted, computer 704 controls monitor716. Additionally, computer 704 is connected to and directs additionalcomponents such as printers, electronic data storage devices and thelike.

Assay Kits

The present invention also provides kits for utilizing the microfluidicdevices of the invention to perform pulsed field mobility shift assays,etc. In particular, these kits typically include microfluidic devices,systems, modules and workstations, etc. A kit optionally containsadditional components for the assembly and/or operation of a multimoduleworkstation of the invention including, but not restricted to roboticelements (e.g., a track robot, a robotic armature, or the like), platehandling devices, fluid handling devices, and computers (including e.g.,input devices, monitors, c.p.u., system software to analyze mobilityshift data from pulsed field assays, and the like).

Generally, the microfluidic devices described herein are optionallypackaged to include some or all reagents for performing the device'sfunctions (e.g., the reagents used in pulsed field assays). For example,the kits can optionally include any of the microfluidic devicesdescribed along with assay components, buffers, reagents, enzymes, serumproteins, receptors, sample materials, antibodies, substrates, controlmaterial, spacers, buffers, immiscible fluids, etc., for performing thepulsed field assays as well as other ancillary and/or additional actionssuch as separations, etc. using the methods/devices of the invention. Inthe case of prepackaged reagents, the kits optionally includepre-measured or pre-dosed reagents that are ready to incorporate intothe assays without measurement, e.g., pre-measured fluid aliquots, orpre-weighed or pre-measured solid reagents that can be easilyreconstituted by the end-user of the kit.

Such kits also typically include appropriate instructions for using thedevices and reagents, and in cases where the reagents (or all necessaryreagents) are not predisposed in the devices themselves, withappropriate instructions for introducing the reagents into thechannels/chambers/reservoirs/etc. of the device. In this latter case,these kits optionally include special ancillary devices for introducingmaterials into the microfluidic systems, e.g., appropriately configuredsyringes/pumps, or the like (in some embodiments, the device itselfcomprises a pipettor element, such as an electropipettor for introducingmaterial into channels/chambers/reservoirs/etc. within the device). Inthe former case, such kits typically include a microfluidic device withnecessary reagents predisposed in the channels/chambers/reservoirs/etc.of the device. Generally, such reagents are provided in a stabilizedform, so as to prevent degradation or other loss during prolongedstorage, e.g., from leakage. A number of stabilizing processes arewidely used for reagents that are to be stored, such as the inclusion ofchemical stabilizers (e.g., enzymatic inhibitors,microbicides/bacteriostats, anticoagulants, etc.), the physicalstabilization of the material, e.g., through immobilization on a solidsupport, entrapment in a matrix (e.g., a bead, a gel, etc.),lyophilization, or the like.

The elements of the kits of the present invention are typically packagedtogether in a single package or set of related packages. The packageoptionally includes written instructions for utilizing one or moredevice of the invention, e.g., primarily the pulsed field assays, inaccordance with the methods described herein. Kits also optionallyinclude packaging materials or containers for holding the microfluidicdevice, system or reagent elements.

The discussion above is generally applicable to the aspects andembodiments of the invention described herein. Moreover, modificationsare optionally made to the methods and devices described herein withoutdeparting from the spirit and scope of the invention as claimed, and theinvention is optionally put to a number of different uses including thefollowing:

The use of a microfluidic system containing at least a first substrateand having a first channel and a second channel intersecting the firstchannel, at least one of the channels having at least onecross-sectional dimension in a range from 0.1 to 500 micrometer, inorder to test the effect of each of a plurality of test compounds on abiochemical system through use of a pulsed field mobility shift assay.

The use of a microfluidic system as described herein, wherein abiochemical system flows through one of said channels substantiallycontinuously, providing for, e.g., sequential testing of a plurality oftest compounds. Such as continuous pulsed field assays.

The use of a microfluidic device as described herein to monitorreactions within microchannels/microchambers/reservoirs/etc.

The use of electrokinetic injection in a microfluidic device asdescribed herein to modulate or achieve flow in the channels.

The use of a combination of wicks, hydrostatic pressure, electrokineticinjection and pressure based flow elements in a microfluidic device asdescribed herein to modulate, focus, or achieve flow of materials, e.g.,in the channels of the device.

The use of pulsed field electrokinetic flow to achieve separation ofmolecules based upon their respective electrophoretic mobility.

An assay utilizing a use of any one of the microfluidic systems orsubstrates described herein.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application, orother document were individually indicated to be incorporated byreference for all purposes.

1. A method of detecting an interaction between a first molecule and atleast a second molecule in a microfluidic device, the method comprising:a) flowing a first labeled molecule with a first electrophoreticmobility, through at least a first microchannel; b) flowing at least asecond molecule with a second electrophoretic mobility through themicrochannel; c) applying a pulsed electric field through themicrochannel, wherein the pulsed electric field separates the first andat least second molecule based upon the electrophoretic mobilities ofthe molecules; and, d) detecting a level of label or a level of signalfrom a label in the microchannel over a select time period.
 2. Themethod of claim 1, wherein the at least second molecule comprises asecond molecule and at least a third molecule.
 3. The method of claim 2,wherein the first molecule comprises a first component of areceptor-ligand pair, the second molecule comprises a second componentof the receptor-ligand pair, and the at least third molecule comprisesthe receptor-ligand pair.
 4. The method of claim 2, wherein the firstmolecule comprises a first component of an antibody-antigen pair, thesecond molecule comprises a second component of the antibody-antigenpair, and the at least third molecule comprises the antibody-antigenpair.
 5. The method of claim 2, wherein the first molecule comprises afirst component of an at least partially complementary double-strandednucleic acid, the second molecule comprises a second component of the atleast partially complementary double-stranded nucleic acid, and the atleast third molecule comprises a double-stranded nucleic acid comprisingthe first and second components.
 6. The method of claim 2, wherein thefirst molecule comprises a substrate, the second molecule comprises anenzyme and the at least third molecule comprises a product.
 7. Themethod of claim 6, wherein the product comprises the label of the firstmolecule.
 8. The method of claim 6, wherein the product comprises thefirst molecule, wherein such molecule comprises a changedelectrophoretic mobility.
 9. The method of claim 1, wherein the at leastsecond molecule comprises a derivative form of the first molecule. 10.The method of claim 2, further comprising at least a fourth molecule,wherein the fourth molecule comprises one or more of: a reactionenhancer, a reaction inhibitor, or a reaction competitor.
 11. The methodof claim 2, wherein a size of one or more of: the first molecule, thesecond molecule, the third molecule, or the fourth molecule changesafter the interaction.
 12. The method of claim 2, wherein a charge ofone or more of: the first molecule, the second molecule, the thirdmolecule, or the fourth molecule, changes after the interaction.
 13. Themethod of claim 2, wherein an electrophoretic mobility of one or moreof: the first molecule, the second molecule, the third molecule, or thefourth molecule, changes after the interaction.
 14. The method of claim1, wherein the interaction comprises one or more of: an enzymaticreaction or a binding reaction.
 15. The method of claim 1, whereinflowing comprises use of one of more of: electrophoretic transport,electroosmotic transport, pressure based transport, wicking basedtransport, or hydrostatic pressure based transport.
 16. The method ofclaim 15, wherein flowing comprises an optimization of a measurement ofassay sensitivity.
 17. The method of claim 16, wherein optimization of ameasurement of assay sensitivity comprises creation of a greatestdifference in one or more reaction parameter, wherein such parameter iscompared between when the pulsed electric field is on and when thepulsed electric field is off.
 18. The method of claim 1, wherein flowingcomprises incubating the first and at least second molecule together fora specific period of time.
 19. The method of claim 1, wherein flowing ofthe first molecule comprises a continuous injection of the firstmolecule into the microchannel and flowing of the second moleculecomprises an intermittent injection of the at least second molecule intothe microchannel.
 20. The method of claim 19, wherein the intermittentinjection of the at least second molecule comprises a first time periodand wherein the pulsed electric field comprises a second time period andwherein the first period is at least as long as the second period. 21.The method of claim 1, wherein flowing of the first molecule comprises acontinuous injection of the first molecule into the microchannel andflowing of the second molecule comprises a continuous injection of theat least second molecule into the microchannel.
 22. The method of claim1, wherein flowing of the first molecule comprises an intermittentinjection of the first molecule into the microchannel and flowing of thesecond molecule comprises an intermittent injection of the at leastsecond molecule into the microchannel.
 23. The method of claim 22,wherein the intermittent injection of the first molecule and theintermittent injection of the second molecule comprise partially orcompletely concurrent injections into the microchannel.
 24. The methodof claim 1, wherein flowing of the first molecule and flowing of the atleast second molecule is concurrent.
 25. The method of claim 1, whereinflowing of the first molecule and flowing of the at least secondmolecule is non-concurrent.
 26. The method of claim 1, wherein the labelcomprises one or more of: a fluorescent label, a chemiluminescent label,or a radioactive label.
 27. The method of claim 1, wherein theelectrophoretic mobility of the first molecule is greater than theelectrophoretic mobility of the at least second molecule.
 28. The methodof claim 1, wherein the electrophoretic mobility of the first moleculeis lesser than the electrophoretic mobility of the at least secondmolecule.
 29. The method of claim 1, wherein the at least secondmolecule comprises a label.
 30. The method of claim 29, wherein thelabel of the at least second molecule comprises a same label type as thelabel of the first molecule.
 31. The method of claim 29, wherein thelabel of the at least second molecule comprises a different label typethan the label of the first molecule.
 32. The method of claim 1, whereinapplying a pulsed electric field comprises applying a first specificvoltage or electric current through the microchannel for a firstspecific period of time followed by applying a second specific voltageor electric current for a second specific period of time.
 33. The methodof claim 32, wherein the first and second periods of time are equal. 34.The method of claim 33, wherein the first and second time periodscomprise from at least about 0.1 second to about 20 seconds or more,from at least about 0.5 seconds to about 15 seconds or more, from atleast 1 second to at least about 10 seconds or more, or from at leastabout 5 seconds to at least about 7 seconds or more.
 35. The method ofclaim 32, wherein the first and second periods of time are not equal.36. The method of claim 35, wherein the first and second time perioddiffer by a factor of about 1, of about 2, of about 3, of about 4, ofabout 5, of about 10, of about 25, or of about
 50. 37. The method ofclaim 32, wherein the first and second specific voltages or electriccurrents comprise from at least about 10 V/cm to about 3,000 V/cm ormore, from at least about 50 V/cm to about 2,000 V/cm or more, from atleast about 100 V/cm to at least about 1,000 V/cm or more, or from atleast about 250 V/cm to at least about 750 V/cm or more.
 38. The methodof claim 32, wherein flowing of the first molecule comprises acontinuous injection of the first molecule into the microchannel andflowing of the second molecule comprises an intermittent injection ofthe at least second molecule into the microchannel.
 39. The method ofclaim 38, wherein the first and second specific periods of time areselectively set in relation to the flowing of the second molecule intothe microchannel and wherein the flowing of the second molecule into themicrochannel comprises a third specific period of time.
 40. The methodof claim 39, wherein the first or second period of time is from at leastabout 1 to about 100 or more, from at least about 5 to at least about75, from at least about 10 to at least about 50, or from at least about25 to at least about 45 times shorter than the third period of time. 41.The method of claim 1, wherein detecting comprises determining reactionkinetics for the interaction between the first molecule and the at leastsecond molecule.
 42. The method of claim 1, wherein the at least firstmicrochannel comprises one or more of: a polymer gel, a microfabricatedbarrier, or a sieving matrix.